What is TempO-Seq™?

Templated Oligo assay with Sequencing readout (TempO-Seq™) is a ligation-based targeted whole transcriptome expression profiling assay designed to maximize the utilization of precious or limited samples for high-throughput toxicogenomic or drug discovery pipelines (Yeakley et al., 2017).

TempO-Seq™ technology harnesses the power of sample barcoding combined with targeted oligos to allow simultaneous measurements of the expression of hundreds to thousands of genes in up to 6,144 samples in the same sequencing run.

In this article, we discuss the technology behind TempO-Seq™ and how its transcriptomic read-outs can accelerate drug discovery with challenging sample types.

TempO-Seq™ is RNA-extraction-free and hybridization-based

Traditional RNA-seq technologies often rely on time-consuming and costly nucleic acid purification, reverse transcription of total RNA to cDNA, and pre-amplification of RNA to increase the number of reads when quantities of RNA are low. But each of these processes loses efficiency and struggles to provide accurate and reliable data with low input or degraded RNA common to formalin-fixed paraffin-embedded (FFPE) samples, tissue microarrays, or liquid biopsies routinely available for large-scale studies.

To avoid these problematic RNA purification, reverse transcription, and amplification stages, TempO-Seq™ works directly with cell lysates and is hybridization-based with a next-generation sequencing read-out. This allows researchers to perform cost-effective transcriptomics on precious samples with as little as 10 pg of total RNA (the amount from a single cell) or degraded RNA (down to RIN 1-3), making TempO-Seq™ ideal for high-quality gene expression profiling of precious biological samples or small, pre-defined areas on an FFPE slide (Trejo et al., 2019).

Improvements to a previous hybridization-based method

TempO-Seq™ achieves this level of sensitivity thanks to some innovative improvements to a different targeted oligo hybridization approach called RASL-seq.

Both technologies use two detector oligos designed to hybridize to adjacent target sequences in a given transcript. When both oligos are properly hybridized to the correct target locations, the two detector oligos are ligated, excess unhybridized oligos are removed, and the ligated oligo pairs are amplified to add sample-specific barcodes and sequencing adapters. Sample barcodes permit the pooling of all samples, removing the need to prepare each sample individually and reducing data variability, cost, and hands-on time.

With RASL-seq, the design of these detector oligos is restricted to splice junctions at the 3’ end of transcripts, limiting the efficiency of hybridization and leading to suboptimal sequence-dependent ligation efficiencies that compromise assay robustness. In TempO-Seq™ detector oligos are designed to maximize hybridization specificity to each gene instead of being limited to 3’ splice junctions (Yeakley et al 2017). The RNA preparation workflow can be completed in six hours with one hour of hands-on time.

TempO-Seq™ also improves throughput compared to RASL-seq and measures a specific sequence within around 20,000 genes in the transcriptome or users can design fully customizable detector oligo panels. Up to 6,144 samples can be pooled in the same sequencing run. In contrast, RASL-seq is limited to profiling a maximum of 500 genes in 1,536 samples simultaneously but excels at detecting splice junctions.

Uses in drug discovery and for challenging sample types

Researchers have successfully used transcriptomic profiles generated by TempO-Seq™ to classify compounds, identify therapeutic efficacy or indicate compound toxicity, and differentiate between mechanisms of action of similar compounds (Nunes et al., 2022; Harrill et al., 2021; Canizzo et al., 2022).

Furthermore, TempO-Seq™ outperforms traditional RNA-seq methods when used to generate gene expression profiles from over 20-year-old FFPE samples with highly degraded RNA (Cannizzo et al., 2022). As FFPE tissue is often the only available biological archive for many toxicological and clinical studies, sensitive techniques such as TempO-seq™ stand to advance our understanding of disease and the effects of compound treatments in samples where robust data generation was previously very challenging.

To learn more about TempO-Seq™ or other ultra-high-throughput transcriptome profiling methods, don't hesitate to get in touch with us by sending an email here.

References

• Cannizzo, M.D. et al. (2022) ‘Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action’, Toxicology Reports, 9, pp.883-894. Available at: https://doi.org/10.1016/j.toxrep.2022.04.012.

• Harrill, J.A. et al. (2021) ‘High-throughput transcriptomics platform for screening environmental chemicals’, Toxicological Sciences, 181(1), pp.68-89. https://doi.org/10.1093/toxsci/kfab009.

• Nunes, C. et al. (2022) ‘An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat’, Toxicology in Vitro, 81, p.105333. Available at: https://doi.org/10.1016/j.tiv.2022.105333.

• Trejo, C.L. et al. (2019) ‘Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue’, PLoS One, 14(2), p.e0212031. Available at: https://doi.org/10.1371/journal.pone.0212031.

• Yeakley, J.M. et al. (2017) ‘A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling’, PloS one, 12(5), p.e0178302. Available at: https://doi.org/10.1371/journal.pone.0178302.