TempO-seq and RNA-seq gene expression levels are highly correlated for most genes: A comparison using 39 human cell lines

Laura J Word, Clinton M Willis, Richard S Judson, Logan J Everett, Sarah E Davidson-Fritz, Derik E Haggard, Bryant A Chambers, Jesse D Rogers, Joseph L Bundy, Imran Shah, Nisha S Sipes and Joshua A Harrill

PLOS ONE, 2025, vol. 20, issue 5, 1-22

Abstract: Recent advances in transcriptomics technologies allow for whole transcriptome gene expression profiling using targeted sequencing techniques, which is becoming increasingly popular due to logistical ease of data acquisition and analysis. As data from these targeted sequencing platforms accumulates, it is important to evaluate their similarity to traditional whole transcriptome RNA-seq. Thus, we evaluated the comparability of TempO-seq data from cell lysates to traditional RNA-Seq from purified RNA using baseline gene expression profiles. First, two TempO-seq data sets that were generated several months apart at different read depths were compared for six human cell lines. The average Pearson correlation was 0.93 (95% CI: 0.90–0.96) and principal component analysis (PCA) showed that these two TempO-seq data sets were highly reproducible and could be combined. Next, TempO-seq data was compared to RNA-Seq data for 39 human cell lines. The log2 normalized expression data for 19,290 genes within both platforms were well correlated between TempO-seq and RNA-seq (Pearson correlation 0.77, 95% CI: 0.76–0.78), and the majority of genes (15,480 genes, 80%) had concordant gene expression levels. PCA showed a platform divergence, but this was readily resolved by calculating relative log2 expression (RLE) of genes compared to the average expression across cell lines in each platform. Application of gene ontology analysis revealed that ontologies associated with histone and ribosomal functions were enriched for the 20% of genes with non-concordant expression levels (3,810 genes). On the other hand, gene ontologies annotated to cellular structure functions were enriched for genes with concordant expression levels between the platforms. In conclusion, we found TempO-seq baseline expression data to be reproducible at different read depths and found TempO-seq RLE data from lysed cells to be comparable to RNA-seq RLE data from purified RNA across 39 cell lines, even though the datasets were generated by different laboratories using different cell stocks.

Date: 2025
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DOI: 10.1371/journal.pone.0320862

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