A comparison of the TempO-Seq and Affymetrix microarray platform using RTqPCR validation

Abstract

Next-generation risk assessment relies on mechanistic data from new approach methods, including transcriptome data. Various technologies, such as high-throughput targeted sequencing methods and microarray technologies based on hybridization with complementary probes, are used to determine differentially expressed genes (DEGs). The integration of data from different technologies requires a good understanding of the differences arising from the use of various technologies.To better understand the differences between the TempO-Seq platform and Affymetrix chip technology, whole-genome data for the volatile compound dimethylamine were compared. Selected DEGs were also confirmed using RTqPCR validation. Although the overlap of DEGs between TempO-Seq and Affymetrix was no higher than 37%, a comparison of the gene regulation in terms of log2fold changes revealed a very high concordance. RTqPCR confirmed the majority of DEGs from either platform in the examined dataset. Only a few conflicts were found (11%), while 22% were not confirmed, and 3% were not detected.Despite the observed differences between the two platforms, both can be validated using RTqPCR. Here we highlight some of the differences between the two platforms and discuss their applications in toxicology.

Keywords: Affymetrix; Chemical perturbation; Microarray; RT2 profiling arrays; RTqPCR; TempOSeq; Transcriptomics.

Fig. 1

Principal component analysis (PCA) shows a separation of samples that correlates with their respective concentration levels: high dose (HD), mid dose (MD), low dose (LD), clean air control (CA), and untreated (UT). For the labelling of the individual samples a table is included in Supplement 2. One sample (S_6_B5_3) from the TempO-Seq mid dose group was removed as an outlier, due to its large distance from other samples. The outlier is not included in the plot for better visibility of the remaining samples in the PCA. See the Supplement 3 for PCA with outlier sample included. The clusters formed by the treatment conditions, (colored circles) show more overlap for the microarray experiment (A) compared to TempO-Seq (B). This indicates that the treatment-related differences among the samples are less pronounced in the microarray experiment

Fig. 2

Scatterplot of the log2 transformed fold changes for genes differentially expressed in Affymetrix and/or TempO-Seq at the HD: red – DEG in Affymetrix only; blue – DEG in TempO-Seq only, green – DEG in both platforms. DEGs significant in both platforms (green) show consistency in terms of direction. Whereas some DEGs of either platform (red & blue) can be found expressed in opposing direction (top-left, bottom-right) albeit not being significant for one platform or direction

Fig. 3

The heatmap shows the log2 fold changes for 238 genes, which have been shown to be differentially expressed in either the Affymetrix or TempO-Seq platforms. Generally, both platforms show similar expression direction with the lower dose showing little change overall with 28 genes being significantly differentially expressed

Fig. 4

The scatterplots show DEGs of the two platforms for Affymetrix (n = 120) and TempO-Seq (n = 205) stratified for being DEG at HD in RTqPCR platform. The individual panels show (A) Affymetrix DEGs with significance in RTqPCR, (B) TempO-Seq DEGs with significance in RTqPCR, (C) Affymetrix DEGs without significance in RTqPCR, and (D) TempO-Seq DEGs without significance in RTqPCR validation. Correlation scores are shown as R in the scatterplots. Correlations between the fold changes of the individual platform with the validation dataset of 238 genes comparing the two test platforms

Fig. 5

Comparison of individual gene expression for a selection of genes between all systems. The bars represent the log2 fold change for selected genes at LD, MD and HD. This selection exemplifies confirmation of DEGs using RTqPCR as well as conflicting results. Padj refers to p-value for Affymetrix and RTqPCR and to FDR for TempO-Seq (< 0.05)

Fig. 6

Individual transcripts of DCLK1 and alignment sites of TempO-Seq probes DCLK1_27096 and DCLK1_20678. The Affymetrix probes for DCLK1 were combined on one row at under TC1300008609.hg.1. Some Affymetrix probes align to multiple sites within the DCLK1 gene, the first occurrence per probe is indicated in the plot. The current version (.5) of ENST00000615680 is no longer covered by the TempO-Seq probe