Excess False Positive Rates in Methods for Differential Gene Expression Analysis using RNA-Seq Data

David M Rocke, Luyao Ruan, Yilun Zhang, J. Jared Gossett, Blythe Durbin-Johnson, Sharon Aviran
doi: http://dx.doi.org/10.1101/020784

Motivation: An important property of a valid method for testing for differential expression is that the false positive rate should at least roughly correspond to the p-value cutoff, so that if 10,000 genes are tested at a p-value cutoff of 10−4, and if all the null hypotheses are true, then there should be only about 1 gene declared to be significantly differentially expressed. We tested this by resampling from existing RNA-Seq data sets and also by matched negative binomial simulations. Results: Methods we examined, which rely strongly on a negative binomial model, such as edgeR, DESeq, and DESeq2, show large numbers of false positives in both the resampled real-data case and in the simulated negative binomial case. This also occurs with a negative binomial generalized linear model function in R. Methods that use only the variance function, such as limma-voom, do not show excessive false positives, as is also the case with a variance stabilizing transformation followed by linear model analysis with limma. The excess false positives are likely caused by apparently small biases in estimation of negative binomial dispersion and, perhaps surprisingly, occur mostly when the mean and/or the dis-persion is high, rather than for low-count genes.

Resolving microsatellite genotype ambiguity in populations of allopolyploid and diploidized autopolyploid organisms using negative correlations between alleles

Lindsay V Clark, Andrea Drauch Schreier
doi: http://dx.doi.org/10.1101/020610

A major limitation in the analysis of genetic marker data from polyploid organisms is non-Mendelian segregation, particularly when a single marker yields allelic signals from multiple, independently segregating loci (isoloci). However, with markers such as microsatellites that detect more than two alleles, it is sometimes possible to deduce which alleles belong to which isoloci. Here we describe a novel mathematical property of codominant marker data when it is recoded as binary (presence/absence) allelic variables: under random mating in an infinite population, two allelic variables will be negatively correlated if they belong to the same locus, but uncorrelated if they belong to different loci. We present an algorithm to take advantage of this mathematical property, sorting alleles into isoloci based on correlations, then refining the allele assignments after checking for consistency with individual genotypes. We demonstrate the utility of our method on simulated data, as well as a real microsatellite dataset from a natural population of octoploid white sturgeon (Acipenser transmontanus). Our methodology is implemented in the R package polysat version 1.4.

Are Genetic Interactions Influencing Gene Expression Evidence for Biological Epistasis or Statistical Artifacts?

Alexandra Fish, John A. Capra, William S Bush
doi: http://dx.doi.org/10.1101/020479

Interactions between genetic variants, also called epistasis, are pervasive in model organisms; however, their importance in humans remains unclear because statistical interactions in observational studies can be explained by processes other than biological epistasis. Using statistical modeling, we identified 1,093 interactions between pairs of cis-regulatory variants impacting gene expression in lymphoblastoid cell lines. Factors known to confound these analyses (ceiling/floor effects, population stratification, haplotype effects, or single variants tagged through linkage disequilibrium) explained most of these interactions. However, we found 15 interactions robust to these explanations, and we further show that despite potential confounding, interacting variants were enriched in numerous regulatory regions suggesting potential biological importance. While genetic interactions may not be the true underlying mechanism of all our statistical models, our analyses discover new signals undetected in standard single-marker analyses. Ultimately, we identified new complex genetic architectures regulating 23 genes, suggesting that single-variant analyses may miss important modifiers.

The weighting is the hardest part: on the behavior of the likelihood ratio test and score test under weight misspecification in rare variant association studies

Camelia Claudia Minica, Giulio Genovese, Dorret I. Boomsma, Christina M. Hultman, René Pool, Jacqueline M. Vink, Conor V. Dolan, Benjamin M. Neale
doi: http://dx.doi.org/10.1101/020198

Rare variant association studies are gaining importance in human genetic research with the increasing availability of exome/genome sequence data. One important test of association between a target set of rare variants (RVs) and a given phenotype is the sequence kernel association test (SKAT). Assignment of weights reflecting the hypothesized contribution of the RVs to the trait variance is embedded within any set-based test. Since the true weights are generally unknown, it is important to establish the effect of weight misspecification in SKAT. We used simulated and real data to characterize the behavior of the likelihood ratio test (LRT) and score test under weight misspecification. Results revealed that LRT is generally more robust to weight misspecification, and more powerful than score test in such a circumstance. For instance, when the rare variants within the target were simulated to have larger betas than the more common ones, incorrect assignment of equal weights reduced the power of the LRT by ~5% while the power of score test dropped by ~30%. Furthermore, LRT was more robust to the inclusion of weighed neutral variation in the test. To optimize weighting we proposed the use of a data-driven weighting scheme. With this approach and the LRT we detected significant enrichment of case mutations with MAF below 5% (P-value=7E-04) of a set of highly constrained genes in the Swedish schizophrenia case-control cohort of 4,940 individuals with observed exome-sequencing data. The score test is currently widely used in sequence kernel association studies for both its computational efficiency and power. Indeed, assuming correct specification, in some circumstances the score test is the most powerful test. However, our results showed that LRT has the compelling qualities of being generally more robust and more powerful under weight misspecification. This is a paramount result, given that, arguably, misspecified models are likely to be the rule rather than the exception in the weighting-based approaches.

The next 20 years of genome research

Michael Schatz
doi: http://dx.doi.org/10.1101/020289

The last 20 years have been a remarkable era for biology and medicine. One of the most significant achievements has been the sequencing of the first human genomes, which has laid the foundation for profound insights into human genetics, the intricacies of regulation and development, and the forces of evolution. Incredibly, as we look into the future over the next 20 years, we see the very real potential for sequencing more than one billion genomes, bringing with it even deeper insights into human genetics as well as the genetics of millions of other species on the planet. Realizing this great potential, though, will only be achieved through the integration and development of highly scalable computational and quantitative approaches can keep pace with the rapid improvements to biotechnology. In this perspective, we aim to chart out these future technologies, anticipate the major themes of research, and call out the challenges ahead. One of the largest shifts will be in the training used to prepare the class of 2035 for their highly interdisciplinary world.