BackgroundSleep is a highly conserved behavior, yet its duration and pattern vary extensively among species and between individuals within species. The genetic basis of natural variation in sleep remains unknown.ResultsWe used the Drosophila Genetic Reference Panel (DGRP) to perform a genome-wide association (GWA) study of sleep in D. melanogaster. We identified candidate single nucleotide polymorphisms (SNPs) associated with differences in the mean as well as the environmental sensitivity of sleep traits; these SNPs typically had sex-specific or sex-biased effects, and were generally located in non-coding regions. The majority of SNPs (80.3%) affecting sleep were at low frequency and had moderately large effects. Additive models incorporating multiple SNPs explained as much as 55% of the genetic variance for sleep in males and females. Many of these loci are known to interact physically and/or genetically, enabling us to place them in candidate genetic networks. We confirmed the role of seven novel loci on sleep using insertional mutagenesis and RNA interference.ConclusionsWe identified many SNPs in novel loci that are potentially associated with natural variation in sleep, as well as SNPs within genes previously known to affect Drosophila sleep. Several of the candidate genes have human homologues that were identified in studies of human sleep, suggesting that genes affecting variation in sleep are conserved across species. Our discovery of genetic variants that influence environmental sensitivity to sleep may have a wider application to all GWA studies, because individuals with highly plastic genotypes will not have consistent phenotypes.

BackgroundA generally accepted approach to the analysis of RNA-Seq read count data does not yet exist. We sequenced the mRNA of 726 individuals from the Drosophila Genetic Reference Panel in order to quantify differences in gene expression among single flies. One of our experimental goals was to identify the optimal analysis approach for the detection of differential gene expression among the factors we varied in the experiment: genotype, environment, sex, and their interactions. Here we evaluate three different filtering strategies, eight normalization methods, and two statistical approaches using our data set. We assessed differential gene expression among factors and performed a statistical power analysis using the eight biological replicates per genotype, environment, and sex in our data set.ResultsWe found that the most critical considerations for the analysis of RNA-Seq read count data were the normalization method, underlying data distribution assumption, and numbers of biological replicates, an observation consistent with previous RNA-Seq and microarray analysis comparisons. Some common normalization methods, such as Total Count, Quantile, and RPKM normalization, did not align the data across samples. Furthermore, analyses using the Median, Quantile, and Trimmed Mean of M-values normalization methods were sensitive to the removal of low-expressed genes from the data set. Although it is robust in many types of analysis, the normal data distribution assumption produced results vastly different than the negative binomial distribution. In addition, at least three biological replicates per condition were required in order to have sufficient statistical power to detect expression differences among the three-way interaction of genotype, environment, and sex.ConclusionsThe best analysis approach to our data was to normalize the read counts using the DESeq method and apply a generalized linear model assuming a negative binomial distribution using either edgeR or DESeq software. Genes having very low read counts were removed after normalizing the data and fitting it to the negative binomial distribution. We describe the results of this evaluation and include recommended analysis strategies for RNA-Seq read count data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2353-z) contains supplementary material, which is available to authorized users.