What is DRUG-seq and what are its benefits in drug discovery?

DRUG-seq is a cost-effective, high-throughput RNA-seq method for drug discovery (Ye et al., 2018; Li et al., 2022). To understand the possible effects of a drug treatment on cells, Digital RNA with pertUrbation of Genes (DRUG-seq) uses the sensitive read-out of gene expression for the whole transcriptome. The low-cost, High-throughput nature of this and other novel technologies makes it possible to screen larger sets of compounds under more experimental conditions than ever before.

In this article we highlight DRUG-seq and its key benefits in drug discovery.

What is DRUG-seq?

RNA-seq based methods are powerful tools for drug discovery, but standard RNA-seq library preparation is expensive and labour intensive.

DRUG-seq minimizes costly library preparation. It uses a multiplexing strategy in a fully automated, high-throughput screening pipeline. This leads to significant reductions in cost and hands-on-time.

In the first steps of DRUG-seq, researchers grow cells, treat them with drugs of interest under different conditions, and lyse them in 384 or 1536 well plates.

Secondly, the researcher adds specific reverse transcription primers containing a well ‘barcode’ and a unique molecular identifier (UMI) to each well. Next, mRNA is reverse transcribed and the barcode is incorporated into cDNA via template switching.

The well barcodes and UMIs tag the 3’ end of each mRNA molecule. This means that all samples and cDNAs are uniquely labelled and can be multiplexed from this point onwards. This unique barcode also allows for subsequent demultiplexing of samples and correction for PCR duplicates during final data analysis.

Thanks to this sample pooling, the experiment requires the preparation of only a single library for hundreds of bulk RNA samples from cells treated with different drugs and experimental conditions.

Finally, multiplexed samples are pre-amplified by PCR and undergo ‘tagmentation’ to add index adapters to all barcoded cDNAs. Illumina sequencing platforms require these indexes as standard. The paired-end libraries are then sequenced and the data is analyzed to identify well position, UMI and transcript information.

Researchers can use the unbiased transcriptomic output from DRUG-seq to find novel mechanisms of action or cluster drugs based on their effect on the transcriptional profile of the treated cells; a prospect which was previously prohibitively expensive. We discussed exciting examples of drug discovery driven by DRUG-seq in another article.

What are its benefits in drug discovery?

There are three key benefits to DRUG-seq in drug discovery: reduced hands-on-time, reduced cost, and the generation of an unbiased profile of the whole transcriptome.

Sample multiplexing is the primary driver of all these benefits, however a much lower sequencing depth also reduces cost and hands-on-time, whilst maintaining data accuracy. For example, Ye et al. detected consistent gene expression when they sequenced DRUG-seq libraries at 2 and 13 million reads per well compared to the conventional 42 million reads per sample (Ye et al., 2018). Furthermore, the number of differentially expressed genes they detected at shallow read depths was similar to deeply sequenced samples when comparing different drug treatments.

Together, these benefits allow the analysis of more samples, drug compounds and experimental conditions in one experiment. It enables the cost-effective, transcriptional profiling of thousands of genes across hundreds to thousands of wells, and is compatible with existing compound screening automations in industrial settings.

Other high-throughput transcriptomic methods for drug discovery

Methods such as Bulk RNA Barcoding and sequencing (BRB-seq) are also extremely well suited to drug screening. BRB-seq has undergone extensive optimization to provide sensitive, accurate data even for low quality RNA; a key concern when using precious human samples such as in many drug discovery applications (Alpern et al., 2018).

BRB-seq also uses 3’ barcoding of mRNA to allow sample multiplexing and lower sequencing depth, to reduce cost and hands-on-time, with no loss in data quality compared to conventional methods (Alpern et al., 2018).

Furthermore, Alithea Genomics has extensively optimized BRB-seq to give the best quality data for different cell types, such as blood cells (Blood cell BRB-seq), with the inclusion of a proprietary globin blocker.

Please contact us at info@alitheagenomics.com to find out more about how BRB-seq could help in your high-throughput drug discovery study.

References

• Alpern, D., Gardeux, V., Russeil, J., Mangeat, B., Meireles-Filho, A.C., Breysse, R., Hacker, D. and Deplancke, B., 2019. BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing. Genome biology, 20(1), pp.1-15.

• Ye, C., Ho, D.J., Neri, M., Yang, C., Kulkarni, T., Randhawa, R., Henault, M., Mostacci, N., Farmer, P., Renner, S. and Ihry, R., 2018. DRUG-seq for miniaturized high-throughput transcriptome profiling in drug discovery. Nature communications, 9(1), pp.1-9.
• Li, J., Ho, D.J., Henault, M., Yang, C., Neri, M., Ge, R., Renner, S., Mansur, L., Lindeman, A., Kelly, B. and Tumkaya, T., 2022. DRUG-seq Provides Unbiased Biological Activity Readouts for Neuroscience Drug Discovery. ACS Chemical Biology. 17(6), pp.1401-1414.