RNA-Seq is a widely used method for studying the behavior of genes under different biological conditions. An essential step in an RNA-Seq study is normalization, in which raw data are adjusted to account for factors that prevent direct comparison of expression measures. Errors in normalization can have a significant impact on downstream analysis, such as inflated false positives in differential expression analysis. An underemphasized feature of normalization is the assumptions on which the methods rely and how the validity of these assumptions can have a substantial impact on the performance of the methods. In this article, we explain how assumptions provide the link between raw RNA-Seq read counts and meaningful measures of gene expression. We examine normalization methods from the perspective of their assumptions, as an understanding of methodological assumptions is necessary for choosing methods appropriate for the data at hand. Furthermore, we discuss why normalization methods perform poorly when their assumptions are violated and how this causes problems in subsequent analysis. To analyze a biological experiment, researchers must select a normalization method with assumptions that are met and that produces a meaningful measure of expression for the given experiment.

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Transcriptional regulatory networks allow bacteria to express proteins only when they are needed. Adaptive hypotheses explaining the evolution of regulatory networks assume that unneeded expression is costly and therefore decreases fitness, but the proximate cause of this cost is not clear. We show that the cost in fitness to Escherichia coli strains constitutively expressing the lactose operon when lactose is absent is associated with the process of making the lac gene products, i.e., associated with the acts of transcription and/or translation. These results reject the hypotheses that regulation exists to prevent the waste of amino acids in useless protein or the detrimental activity of unnecessary proteins. While the cost of the process of protein expression occurs in all of the environments that we tested, the expression of the lactose permease could be costly or beneficial, depending on the environment. Our results identify the basis of a single selective pressure likely acting across the entire E. coli transcriptome.A central tenet of evolutionary biology is that tradeoffs arise as organisms allocate limited resources to various competing traits. For example, the differential allocation of intermediary metabolites drives a trade-off between investment in structures that increase reproduction and structures that increase survival (Harshman and Zera 2007). The allocation of limiting amounts of time shapes patterns of animal behavior (Stephens and Krebs 1986). Differences in the availability of nutrients to plants drive a trade-off between a fast-growing, poorly defended strategy and a slow-growing, well-defended strategy (Coley et al. 1985). In all of these cases, tradeoffs shape patterns of morphological and behavioral diversity.Trade-offs occur not only for morphology and behavior, but also for cellular processes such as gene expression. Regulatory networks to control gene expression in response to specific environmental cues have presumably been selected to manage these trade-offs. Experimental evolution can be used to measure the costs of unnecessary expression and to explore the proximate causes of this selection. Zamenhoff and Eichhorn (1967) demonstrated that it is costly for Bacillus subtlis to produce the proteins for trytophan biosynthesis when they are not needed, but did not determine why. Dykhuizen (1978) demonstrated that the cost for Escherichia coli of expressing the same proteins could not be explained by a simple energy conservation argument. Dykhuizen was, however, unable to determine the source of the cost. Several authors (Novick and Weiner 1957;Andrews and Hegeman 1976;Dykhuizen and Davies 1980;Koch 1983) have shown that constitutive expression of the lac operon in E. coli lowers fitness when there is no lactose present in the environment. While various costs have been suggested, no experiments have directly tested the competing hypotheses. The molecular basis of the cost of unnecessary expression, a potentially major force acting on regulatory networks, remains undiscovered.The...

Populations evolving in constant environments exhibit declining adaptability. Understanding the basis of this pattern could reveal underlying processes determining the repeatability of evolutionary outcomes. In principle, declining adaptability can be due to a decrease in the effect size of beneficial mutations, a decrease in the rate at which they occur, or some combination of both. By evolving Escherichia coli populations started from different steps along a single evolutionary trajectory, we show that declining adaptability is best explained by a decrease in the size of available beneficial mutations. This pattern reflected the dominant influence of negative genetic interactions that caused new beneficial mutations to confer smaller benefits in fitter genotypes. Genome sequencing revealed that starting genotypes that were more similar to one another did not exhibit greater similarity in terms of new beneficial mutations, supporting the view that epistasis acts globally, having a greater influence on the effect than on the identity of available mutations along an adaptive trajectory. Our findings provide support for a general mechanism that leads to predictable phenotypic evolutionary trajectories.