Impact of Sequencing Depth and Library Preparation on Toxicological Interpretation of RNA-Seq Data in a “Three-Sample” Scenario

While
RNA-sequencing (RNA-seq) has emerged as a standard approach
in toxicogenomics, its full potential in gaining underlying toxicological
mechanisms is still not clear when only three biological replicates
are used. This “three-sample” study design is common
in toxicological research, particularly in animal studies during preclinical
drug development. Sequencing depth (the total number of reads in an
experiment) and library preparation are critical to the resolution
and integrity of RNA-seq data and biological interpretation. We used
aflatoxin b1 (AFB1), a model toxicant, to investigate the effect of
sequencing depth and library preparation in RNA-seq on toxicological
interpretation in the “three-sample” scenario. We also
compared different gene profiling platforms (RNA-seq, TempO-seq, microarray,
and qPCR) using identical liver samples. Well-established mechanisms
of AFB1 toxicity served as ground truth for our comparative analyses.
We found that a minimum of 20 million reads was sufficient to elicit
key toxicity functions and pathways underlying AFB1-induced liver
toxicity using three replicates and that identification of differentially
expressed genes was positively associated with sequencing depth to
a certain extent. Further, our results showed that RNA-seq revealed
toxicological insights from pathway enrichment with overall higher
statistical power and overlap ratio, compared with TempO-seq and microarray.
Moreover, library preparation using the same methods was important
to reproducing the toxicological interpretation.