Methods detecting rhythmic gene expression are biologically relevant only for strong signal
Authors: Laloum David , Robinson-Rechavi Marc 
Published: August 2019

The nycthemeral transcriptome embodies all genes displaying a rhythmic variation of their mRNAs periodically every 24 hours, including but not restricted to circadian genes. In this study, we show that the nycthemeral rhythmicity at the gene expression level is biologically functional and that this functionality is more conserved between orthologous genes than between random genes. We used this conservation of the rhythmic expression to assess the ability of seven methods (ARSER, Lomb Scargle, RAIN, JTK, empirical-JTK, GeneCycle, and meta2d) to detect rhythmic signal in gene expression. We have contrasted them to a naive method, not based on rhythmic parameters. By taking into account the tissue-specificity of rhythmic gene expression and different species comparisons, we show that no method is strongly favored. The results show that these methods designed for rhythm detection, in addition to having quite similar performances, are consistent only among genes with a strong rhythm signal. Rhythmic genes defined with a standard p-value threshold of 0.01 for instance, could include genes whose rhythmicity is biologically irrelevant. Although these results were dependent on the datasets used and the evolutionary distance between the species compared, we call for caution about the results of studies reporting or using large sets of rhythmic genes. Furthermore, given the analysis of the behaviors of the methods on real and randomized data, we recommend using primarily ARS, empJTK, or GeneCycle, which verify expectations of a classical distribution of p-values. Experimental design should also take into account the circumstances under which the methods seem more efficient, such as giving priority to biological replicates over the number of time-points, or to the number of time-points over the quality of the technique (microarray vs RNAseq). GeneCycle, and to a lesser extent empirical-JTK, might be the most robust method when applied to weakly informative datasets. Finally, our analyzes suggest that rhythmic genes are mainly highly expressed genes.

Author Summary

To be active, genes have to be transcribed to RNA. For some genes, the transcription rate follows a circadian rhythm with a periodicity of approximately 24 hours; we call these genes “rhythmic”. In this study, we compared methods designed to detect rhythmic genes in gene expression data. The data are measures of the number of RNA molecules for each gene, given at several time-points, usually spaced 2 to 4 hours, over one or several periods of 24 hours. There are many such methods, but it is not known which ones work best to detect genes whose rhythmic expression is biologically functional. We compared these methods using a reference group of evolutionarily conserved rhythmic genes. We compared data from baboon, mouse, rat, zebrafish, fly, and mosquitoes. Surprisingly, no method was particularly effective. Furthermore, we found that only very strong rhythmic signals were relevant with each method. More precisely, when we use a usual cut-off to define rhythmic genes, the group of genes considered as rhythmic contains many genes whose rhythmicity cannot be confirmed to be biologically relevant. We also show that rhythmic genes mainly contain highly expressed genes. Finally, based on our results, we provide recommendations on which methods to use and how, and suggestions for future experimental designs.