Beware of biological variability in your *-Seq experiments

From this excellent paper on biological variability in RNA-Seq experiments (bold highlights are mine):

“Biological variability has important implications for the design, analysis and interpretation of RNA-sequencing experiments. […] If only a few biological replicates are available, it will be impossible to estimate the level of biological variability in expression for each gene in a study. Supplementary Table 1 summarizes a large number of published RNA-sequencing studies over the past three years. In every case, except for the two studies we analyzed here, conclusions were based on a small number (n ≤ 2) of biological replicates. One goal of RNA-sequencing studies may be simply to identify and catalog expression of new or alternative transcripts. However, all of these studies make broader biological statements on the basis of a very small set of biological replicates.

Our analysis has two important implications for studies performed with a small number of biological replicates. First, significant results in these studies may be due to biological variation and may not be reproducible; and second, it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the study populations. These ideas are now widely accepted for DNA microarray experiments, where a large number of biological replicates are now required to justify scientific conclusions. Our analysis suggests that as biological variability is a fundamental characteristic of gene expression, sequencing experiments should be subject to similar requirements.”

If you are doing RNA-Seq, be very vigilant in your experimental design and find a way to incorporate more replicates, even at the expense of testing fewer comparisons.   It’s better to test one comparison (tissue X vs. Y, for example) with 5 or more replicates than to test three comparisons (Tissue X vs. Y, Y vs. Z, and X vx Z) with only 2 replicates for each tissue type.


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