Comparing TempO-seq and RNA-seq mRNA data sets

Poster presented to the Society of Toxicology 62nd Annual Meeting and ToxExpo March 2023

There are multiple technological platforms available for quantifying mRNA levels to use for transcriptomics studies. With the increase in mRNA expression data generated using TempO-seq, it is important to determine whether TempO-seq and RNA-seq data are comparable and/or can be combined.

Researchers at the EPA describe a workflow process for evaluating whether and how mRNA data sets can be combined when comparing and/or aggregating data generated by TempO-seq versus RNA-seq. For this analysis, two different EPA-generated TempO-seq data sets were compared to each other using principal component analysis (PCA) for four overlapping cell types (Daudi, HepG2, MCF-7, and U-2 OS) and the PCA showed that the data sets could be combined. The TempO-seq data were then compared to RNA-seq data for 12 cell types (A549, Daudi, HBEC3-KT, HepG2, HME-1, HUVEC, MCF-7, RPE-1, RPTEC, TIME, U-2 OS, and T47-D) using PCA. For the four overlapping cell types within both EPA-generated TempO-seq data sets, the average of all replicates per cell type were used for this comparison to RNA-seq.

Statistical testing using PCA on TempO-seq versus RNA-seq mRNA expression data for the 19,119 overlapping genes showed that there was a clear platform divergence pattern within the first principal component (PC1) for all cell types evaluated. This meant that these TempO-seq and RNA-seq data should not be combined without further steps. Normalizing the data by calculating the relative log expression (RLE) compared to the average expression level across cell types in each platform removed the platform divergence observed in PC1. Removing genes with large differences in expression levels between the technological platforms was also found to be effective in resolving platform divergence. This work should help increase scientific confidence when using TempO-seq data for conducting transcriptomics research. The views expressed are those of the authors and do not necessarily represent the views or the policies of the U.S. EPA.

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