Strategies for detecting and identifying biological signals amidst the variation commonly found in RNA sequencing data.

backgroundRNA sequencing analysis focus on the detection of differential gene expression changes that meet a two-fold minimum change between groups. The variability present in RNA sequencing data may obscure the detection of valuable information when specific genes within certain samples display large expression variability. This paper develops methods that apply variance and dispersion estimates to intra-group data to identify genes with expression values that diverge from the group envelope. STRING database analysis of the identified genes characterize gene affiliations involved in physiological regulatory networks that contribute to biological variability. Individuals with divergent gene groupings within network pathways can thereby be identified and judiciously evaluated prior to standard differential analysis.

resultsA three-step process is presented for evaluating biological variability within a group in RNA sequencing data in which gene counts were: (1) scaled to minimize heteroscedasticity; (2) rank-ordered to detect potentially divergent "trendlines" for every gene in the data set; and (3) tested with the STRING database to identify statistically significant pathway associations among the genes displaying marked trendline variability and dispersion. This approach was used to identify the "trendline" profile of every gene in three test data sets. Control data from an in-house data set and two archived samples revealed that 65-70% of the sequenced genes displayed trendlines with minimal variation and dispersion across the sample group after rank-ordering the samples; this is referred to as a linear trendline. Smaller subsets of genes within the three data sets displayed markedly skewed trendlines, wide dispersion and variability. STRING database analysis of these genes identified interferon-mediated response networks in 11-20% of the individuals sampled at the time of blood collection. For example, in the three control data sets, 14 to 26 genes in the defense response to virus pathway were identified in 7 individuals at false discovery rates ≤1.92 E-15.

conclusionsThis analysis provides a rationale for identifying and characterizing notable gene expression variability within a study group. The identification of highly variable genes and their network associations within specific individuals empowers more judicious inspection of the sample group prior to differential gene expression analysis.