Microarray-based Clariom™ assays from Applied Biosystems™ are a potential option for gene expression profiling studies when looking for a scalable technology to integrate into drug screening pipelines.
But there are pitfalls to microarray-based technologies, such as pre-selection of probe targets, relatively high cost per sample, a narrower quantitative range of expression level changes, and poorer detection of differentially expressed genes between conditions compared to RNA-seq-based methods (Rao et al., 2019).
In contrast to microarray technologies, the dramatic decrease in the cost of next-generation sequencing (NGS) and the increased throughput now enabled by new 3’ bulk mRNA-seq sample barcoding approaches permit unbiased insight into all transcripts with a wider dynamic range of expression at a lower cost (Alpern et al., 2019; Rao et al., 2019).
Here, we compare the Clariom™ GO Screen to an ultra-scalable 3’ bulk mRNA-seq method called MERCURIUS™ DRUG-seq from Alithea Genomics, which is an attractive alternative to accelerate the development of new drug compounds with robust, scalable transcriptomic insights.
The Clariom™ GO Screen: Pre-selected targets versus unbiased gene expression profiling
Like the Clariom™ S assay, the Clariom™ GO Screen provides gene expression information for around 20,000 human genes, and each array includes probes derived from the Human Gene Ontology gene collection to ensure coverage of literature-associated “biomarker” genes, immunology markers, and inflammation markers (Thermo Fisher Scientific, 2023).
But this limit of 20,000 pre-selected genes represents a potential stumbling block when an unbiased transcriptome-wide view is required and could be especially important for detecting novel transcripts after drug treatment or in response to genomic perturbations.
One solution to this issue is MERCURIUS™ DRUG-seq.
Because the MERCURIUS™ library preparation technology does not rely on pre-selected targets, users have the flexibility to sequence samples to different depths depending on the core goals of a study.
For instance, if researchers want to examine the expression of genes on a global scale or focus on identifying highly expressed genes that may contain novel transcripts, they may opt to sequence samples to a shallow depth of one to five million reads. This read-depth allows users to detect approximately 18,000 genes while enabling robust differential expression analyses between different samples or experimental conditions.
On the other hand, if the aim of a study is to investigate genes of interest with low expression levels, deeper sequencing of MERCURIUS™ DRUG-seq libraries increases the likelihood of their detection with no restriction to known sequences.
This unbiased read-out is possible because MERCURIUS™ DRUG-seq adds sample barcodes and unique molecular identifiers (UMIs) to the 3’ poly-A tail of each mRNA molecule at the reverse transcription step early in the workflow. This facilitates the early pooling of samples into the same tube and allows for the demultiplexing of samples and adjustments for PCR amplification artifacts in the final data analysis to achieve robust and accurate expression levels.
No RNA extraction, lower cost
Cost and hands-on time are some of the most important considerations when a study has thousands of samples, and both can quickly spiral as sample size, number of conditions, drugs, or replicates increase.
A major driver of cost and hands-on time in most transcriptomic techniques is the RNA extraction process required before library preparation. To remove this inefficient step, both the Clarium™ GO Screen and MERCURIUS™ DRUG-seq directly lyse cell samples in 384 well plates and use the lysed material for mRNA target and library preparation.
As a result, the hands-on time for complete MERCURIUS™ DRUG-seq library preparation for one 384-well plate is up to 3 hours 30 minutes. The entire protocol takes up to one day to complete depending on the project size and excluding sequencing time on an Illumina or MGI instrument. A MERCURIUS™ DRUG-seq service is also available where users send cell lysates to Alithea Genomics, making it highly adaptable for integration into drug discovery pipelines.
The Clariom™ GO Screen mRNA target preparation takes around two days and is offered as a service through verified providers (Thermo Fisher Scientific, 2023).
In terms of cost, the Clariom™ GO Screen is a cost-effective option for gene expression profiling with a lower price point than the Illumina TruSeq library preparation.
However, the cost per sample is still significantly higher than MERCURIUS™ DRUG-seq which has a price point as low as $2 per sample, translating to significant savings for large-scale studies.
MERCURIUS™ DRUG-seq is ultra-scalable
Scalability is also an important consideration for large studies.
The Clarium™ GO Screen allows users to perform 384 assays on one array plate, and more plates can be added if more samples are required.
With MERCURIUS™ DRUG-seq, 96 or 384 samples can be multiplexed into the same tube early in the library preparation workflow. Users can prepare as little as one tube to a maximum of 64 individual tubes, each containing 384 samples, for a total of 24,576 samples with one kit.
Ultimately, there are benefits to both methods, but integrating gene expression profiling methods into large compound screening pipelines is undoubtedly an important step to understanding how small molecules affect genes and could contribute to improving human health.
- Alpern, D. et al. (2019) ‘BRB-seq: Ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing’, Genome Biology, 20(1), pp.1-15. Available at: https://doi.org/10.1186/s13059-019-1671-x.
- Clariom™ GO Screen Assay | Thermo Fisher Scientific (2023). Available at: https://www.thermofisher.com/order/catalog/product/952361.
- Rao, M.S. et al. (2019) ‘Comparison of RNA-Seq and microarray gene expression platforms for the toxicogenomic evaluation of liver from short-term rat toxicity studies’. Frontiers in genetics, 9, p.636. Available at: https://doi.org/10.3389/fgene.2018.00636.
- Clariom™ GO Screen | Thermo Fisher Scientific (2023). Available at: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0018542_ClariomGO_Screen384HT_UG.pdf.