The importance of tissue specificity for RNA-seq: highlighting the errors of composite structure extractions

Abstract

Background: A composite biological structure, such as an insect head or abdomen, contains many internal structures with distinct functions. Composite structures are often used in RNA-seq studies, though it is unclear how expression of the same gene in different tissues and structures within the same structure affects the measurement (or even utility) of the resulting patterns of gene expression. Here we determine how complex composite tissue structure affects measures of gene expression using RNA-seq.

Results: We focus on two structures in the honey bee (the sting gland and digestive tract) both contained within one larger structure, the whole abdomen. For each of the three structures, we used RNA-seq to identify differentially expressed genes between two developmental stages, nurse bees and foragers. Based on RNA-seq for each structure-specific extraction, we found that RNA-seq with composite structures leads to many false negatives (genes strongly differentially expressed in particular structures which are not found to be differentially expressed within the composite structure). We also found a significant number of genes with one pattern of differential expression in the tissue-specific extraction, and the opposite in the composite extraction, suggesting multiple signals from such genes within the composite structure. We found these patterns for different classes of genes including transcription factors.

Conclusions: Many RNA-seq studies currently use composite extractions, and even whole insect extractions, when tissue and structure specific extractions are possible. This is due to the logistical difficultly of micro-dissection and unawareness of the potential errors associated with composite extractions. The present study suggests that RNA-seq studies of composite structures are prone to false negatives and difficult to interpret positive signals for genes with variable patterns of local expression. In general, our results suggest that RNA-seq on large composite structures should be avoided unless it is possible to demonstrate that the effects shown here do not exist for the genes of interest.

Figure 1

Total transcriptome size of each structure in nurse and forager honey bees. Genes with RPKM > 10.

Figure 2

Genes present or absent in the abdomen relative to sting gland transcriptome. Number of genes expressed (at greater than 10 RPKM) in the sting gland missing in the abdomen, along with the number of genes missing in the sting gland that were present in the whole abdomen. (+) indicates the gene was present in a transcriptome, while a (-) indicates it was missing. Data are for genes present or absent in nurse transcriptomes.

Figure 3

Genes present or absent in the abdomen relative to digestive tract transcriptome. Number of genes expressed in the digestive tract missing in the abdomen, along with the number of genes missing in the digestive tract that were present in the whole abdomen. (+) indicates the gene was present in a transcriptome, while a (-) indicates it was missing. Data are for genes present or absent in nurse transcriptomes. Only genes with RPKM > 10 included.

Figure 4

Genes with the same or different pattern of expression in composite relative to tissue specific extractions. Number of genes found to be differentially expressed between nurse and forager castes using RNA-seq for both the sting gland and digestive tract that were either found to be not differentially expressed in the corresponding whole abdomen tests, or were differentially expressed but in the opposite direction. Results based on NOISeq analysis.

Figure 5

Distribution of ratios of the expression of each gene in both the sting gland and the digestive tract relative to the whole abdomen. A value of 1 means that the gene’s RPKM value was equal in the tissue specific and the composite extraction, while a value above 1 means that the gene was expressed at a higher rate in the tissue specific extraction and showed dilution in the composite extraction. Genes showing dilution are candidates for specialized function.

Figure 6

Transcription factors with the same or different pattern of expression in composite relative to tissue specific extractions. Number of transcription factors found to be differentially expressed between nurse and forager castes using RNA-seq in both the sting gland and digestive tract that were either found to be not differentially expressed in the corresponding whole abdomen tests, or were differentially expressed but in the opposite direction. Results based on NOISeq analysis.