Abstract
The sequencing depth necessary for documenting differential gene expression using RNA-Seq has been little explored outside of model systems. In particular, the depth required to analyze large-scale patterns of differential transcription factor expression is not known. The goal of the present study is to explore the effectiveness of shallow (relatively low read depth) RNA-Seq. We focus on two tissues in the honey bee: the sting gland and the digestive tract. The sting gland is an experimentally well-understood tissue that we use to benchmark the utility of this approach. We use the digestive tract to test the results obtained with the sting gland, and to conduct RNA-Seq between tissue types. Using a list of experimentally verified genes conferring tissue-specific functions in the sting gland, we show that relatively little read depth is necessary to identify them. We argue that this result should be broadly applicable, since genes important for tissue-specific functions often have robust expression patterns, and because we obtained similar results in our analysis of the digestive tract. Furthermore, we demonstrate that the differential expression of transcription factors, which are transcribed at low levels compared to other genes, can nevertheless often be determined using shallow RNA-Seq. Overall, we find over 150 differentially expressed transcription factors in our tissues at a read depth of only 12 million. This work shows the utility of low-depth sequencing for identifying genes important for tissue-specific functions. It also verifies the often-held belief that transcription factors show low levels of expression, while demonstrating that, in spite of this, they are frequently amenable to shallow RNA-Seq. Our findings should be of benefit to researchers using RNA-Seq in many different biological systems.
Highlights
Next-generation sequencing has greatly expanded our capacity to address fundamental questions in genomics [1,2,3,4]
We determine whether shallow RNA-Seq is capable of identifying as differentially expressed the genes in the sting gland conferring tissue specific functions. Having shown that these key functional genes can be identified with shallow RNA-Seq, we turn to the question of whether such genes would be recognizable as important, were they not already experimentally characterized. We do this in two ways: first, we explore the expression patterns of the key genes to show that they stand out relative to other differentially expressed genes (DEGs), and second, we explore whether the identification of genes important for the tissue specific functions of the digestive tract are amenable to the same approach
While the number of DEGs was lower for the developmental phase comparisons relative to the tissue comparisons, this increasing trend was evident for both
Summary
In spite of the progress of the past several years, there are still basic questions relevant to the use of RNA-Seq that remain unanswered for most organisms. Work on the sequencing depth necessary for identifying differentially expressed genes, for example, has been conducted primarily with mammals, and it is not clear that equal depth is necessary for organisms with simpler transcriptomes [18,24,25]. Quantifying the read depth necessary for RNA-Seq might depend on whether the focal genes show high or low levels of expression. Transcription factors (TFs) are thought to be expressed at relatively low rates, but few studies have documented how low these rates are, and what sequencing depth is necessary to document differential expression in these genes [26,27,28]
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