RNA sequencing (RNA-seq) is a widely used approach to investigate gene expression and increasingly is used in time-course studies to characterize transcriptomic changes over time. Two primary options are available as controls in time-course experiments: samples collected at the first sampling time are used as controls (temporal control, TC) and samples collected in parallel at each individual sampling time are used as controls (biological control, BC). While both approaches are used in experimental studies, we know of no analyses performed to date that directly compare effects of control type choices on identifying differentially expressed genes (DEGs) and subsequent functional analysis. In the current study, we compare experimental results using these different control types for time-course RNA-seq drought stress experiments in two wild grass species in the genus Paspalum. Our results showed BC assemblies gave a higher number of loci in both species. The number of DEGs increased with increasing stress and then decreased dramatically at the recovery time point using both control types. Expression levels of the same DEGs were highly correlated between control types in both species, ranging from r = .653 to r = .852. We also observed similar rank orders of shared enriched Gene Ontology term lists using the two different control types. Collectively, our findings suggest similar results in differential gene expression and functional annotation between control types. The ultimate choice of control type will rely on the experimental length and organism type, with labour time and sequencing costs as additional factors to be considered.

中文翻译：

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比较非模型生物中时间序列RNA测序实验的对照选项：以草为例。

RNA测序（RNA-seq）是研究基因表达的一种广泛使用的方法，并且在时程研究中越来越多地用于表征转录组随时间的变化。在时程实验中有两个主要选项可作为对照：在第一个采样时间收集的样品用作对照（时间对照，TC），在每个单独采样时间并行收集的样品用作对照（生物对照，BC） 。虽然这两种方法都用于实验研究中，但我们知道迄今为止尚无可直接比较对照类型选择对鉴定差异表达基因（DEG）和后续功能分析的影响的分析。在目前的研究中 我们比较了使用这些不同控制类型的实验结果，进行了Paspalum属两种野生草种的时程RNA-seq干旱胁迫实验。我们的结果表明，BC装配体在这两个物种中均提供了更多的基因座。使用两种控制类型，DEG的数量都随着应力的增加而增加，然后在恢复时间点急剧下降。在两个物种的对照类型之间，相同DEG的表达水平高度相关，范围从r = .653到r = .852。我们还观察到了使用两种不同控制类型的共享的丰富基因本体术语列表的相似等级。总体而言，我们的发现表明，在对照类型之间的差异基因表达和功能注释方面，也有相似的结果。控制类型的最终选择将取决于实验长度和生物体类型，