Background: Next-generation sequencing (NGS) has made it possible to determine the sequence and relative abundance of all nucleotides in a biological or environmental sample. A cornerstone of NGS is the quantification of RNA or DNA presence as counts. However, these counts are not counts per se: their magnitude is determined arbitrarily by the sequencing depth, not by the input material. Consequently, counts must undergo normalization prior to use. Conventional normalization methods require a set of assumptions: they assume that the majority of features are unchanged and that all environments under study have the same carrying capacity for nucleotide synthesis. These assumptions are often untestable and may not hold when heterogeneous samples are compared. Results: Methods developed within the field of compositional data analysis offer a general solution that is assumption-free and valid for all data. Herein, we synthesize the extant literature to provide a concise guide on how to apply compositional data analysis to NGS count data. Conclusions: In highlighting the limitations of total library size, effective library size, and spike-in normalizations, we propose the log-ratio transformation as a general solution to answer the question, "Relative to some important activity of the cell, what is changing?"

As the world warms, understanding the fundamental mechanisms available to organisms to protect themselves from thermal stress is becoming ever more important. Heat shock proteins are highly conserved molecular chaperones which serve to maintain cellular processes during stress, including thermal extremes. Developing animals may be particularly vulnerable to elevated temperatures, but the relevance of heat shock proteins for developing altricial birds exposed to a thermal stressor has never been investigated. Here, we sought to test whether three stress-induced genes – HSPD1, HSPA2, HSP90AA1 – and two constitutively expressed genes – HSPA8, HSP90B1 – are upregulated in response to acute thermal shock in zebra finch (Taeniopygia guttata) embryos half-way through incubation. Tested on a gradient from 37.5 ◦C (control) to 45 ◦C, we found that all genes, except HSPD1, were upregulated. However, not all genes initiated upregulation at the same temperature. For all genes, the best fitting model included a correlate of developmental stage that, although it was never significant after multiple-test correction, hints that heat shock protein upregulation might increase through embryonic development. Together, these results show that altricial avian embryos are capable of upregulating a known protective mechanism against thermal stress, and suggest that these highly conserved cellular mechanisms may be a vital component of early developmental protection under climate change.