A core goal of large-scale drug discovery screens is to use the transcriptomic effects of novel compounds on cells as indicators of both drug toxicity and potential mechanisms of action.
But, many studies rely on hundreds to thousands of RNA samples extracted from archived formalin-fixed paraffin-embedded tissue (FFPE) or other chemically fixed samples that provide notoriously degraded RNA unsuitable for standard RNA-seq approaches.
Targeted transcriptomic profiling methods such as Ion AmpliSeq™ go some way to providing gene expression read-outs of pre-selected gene panels for tens of degraded RNA samples, but the novel MERCURIUS™ family of technologies, based on bulk RNA barcoding, now gives users the power to generate unbiased transcriptome-wide data for hundreds of samples simultaneously, regardless of RNA quality.
In this article, we discuss how Ion AmpliSeq™ compares to the MERCURIUS™ suite of technologies regarding workflows, sample requirements, multiplexing capacities, and cost per sample.
Targeted Ion AmpliSeq™
Ion AmpliSeq™ is a targeted PCR-based transcriptome profiling technology, meaning that users are restricted to pre-designed oligonucleotide pairs targeting known sequences (Thermo Fisher Scientific 2023).
The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit does provide oligonucleotides targeting over 20,000 genes, but the targeted nature of this technology makes it unsuitable for unbiased exploratory studies interested in the expression of potentially important novel transcripts as biomarkers of drug efficacy and toxicity, or that might hint at mechanisms of action.
Unbiased MERCURIUS™ technologies
In contrast, all of the MERCURIUS™ family of technologies ─ MERCURIUS™ BRB-seq, MERCURIUS™ DRUG-seq, and MERCURIUS™ FFPE-seq ─ provide unbiased windows into the transcriptome as they are not reliant on probes designed for known transcripts.
Instead, the bulk 3’ mRNA-seq methods, MERCURIUS™ BRB-seq and MERCURIUS™ DRUG-seq, harness highly optimized sample barcodes and unique molecular identifiers to tag the 3’ poly(A) tail of all mRNA molecules during the first-strand synthesis step of cDNA library preparation. This barcoding strategy ensures novel transcripts are detected and generates robust, reliable, and reproducible transcriptome-wide gene expression results comparable to Illumina TruSeq library preparations but at a fraction of the cost.
Importantly, these two MERCURIUS™ technologies provide expression information only for the 3’ end of genes, limiting the exploration of fusion genes, novel isoforms, and alternative splicing events.
Therefore, if a study requires unbiased full-length transcript information for highly degraded RNA, MERCURIUS™ FFPE-seq is the most appropriate option as it includes an initial poly(A) tailing step so that each fragment of total RNA in degraded samples can be barcoded and primed for reverse transcription instead of just the 3’ region of mRNA. This ensures read coverage across the entire length of transcripts, regardless of RNA integrity (RIN) values.
RNA sample requirements
With over a billion FFPE samples stored in biobanks and hospitals worldwide (Blow 2007), the hidden transcriptomic information contained within these is an incredibly rich and relatively untapped source of biological information to inform on countless aspects of health, disease, and drug response.
But, to access this information, researchers must use the appropriate transcriptome profiling method suitable for extracted RNA, which is often of poor quality and low quantity.
For instance, MERCURIUS™ BRB-seq is recommended for a range of 50ng-1μg RNA per sample with RIN ≥6.
For optimal results with MERCURIUS™ FFPE-seq, around 500ng of even heavily degraded RNA with RINs as low as one is recommended as input. These are both ideal options for low-quality RNA samples.
Ion AmpliSeq™ requires at least 10ng of isolated RNA but with the trade-off of limited transcriptome information due to its targeted nature (Thermo Fisher Scientific 2023).
If a study has screened compounds in cells, MERCURIUS™ DRUG-seq has the benefit of being completely RNA-extraction-free, meaning as little as 2000/cells per well can be lysed directly in 384 well plates after experimental treatments, making it an extremely convenient option for large-scale automated screening pipelines.
Multiplexing capacity and cost
For studies with hundreds or thousands of samples, selecting a transcriptome profiling technology with high multiplexing capacity is key to cost and time-efficient data generation.
Ion AmpliSeq™ has a maximum multiplexing capacity of only 16 samples when using the Ion 550™ chip, whereas all MERCURIUS™ technologies allow users to multiplex up to 384 samples in the same library pool very early in the workflow, passing significant reductions in cost and hands-on time to the user with no compromise on data quality.
Also, because of the low multiplexing capacity of Ion AmpliSeq™, the cost per sample is much higher than each of the MERCURIUS™ technologies.
Of the MERCURIUS™ family, MERCURIUS™ DRUG-seq and MERCURIUS™ BRB-seq have the lowest cost per sample. Both of these technologies are highly economical as they are bulk 3’ mRNA-seq-based, so sequencing depths of only five million reads per sample is sufficient to detect around 20,000 genes.
In contrast, MERCURIUS™ FFPE-seq has the same multiplexing capacity but is moderately more expensive than MERCURIUS™ DRUG-seq and MERCURIUS™ BRB-seq because it provides users with unbiased full-length transcriptomic read-outs with a recommended sequencing depth of 20 million reads per sample for optimal results.
A balancing act between samples, cost, and research question
Ultimately, the most appropriate choice of transcriptome profiling technology depends on the number and type of samples, the quality and quantity of starting material, the study budget, and the research question.
Where Ion AmpliSeq™ falls short in cost and multiplexing capacity, the MERCURIUS™ family of technologies provides ample solutions for ultra-high-throughput transcriptomic studies of even the most challenging samples.
To find out more about the MERCURIUS™ family of technologies from Alithea Genomics, get in touch with us.
References
- Blow, N. (2007) ‘Tissue issues’, Nature, 448(7156), pp.959-960. Available at: https://doi.org/10.1038/448959a.
- Thermo Fisher Scientific (2023) ‘Ion AmpliSeq™ Targeted Sequencing Technology’. Available at: https://www.thermofisher.com/uk/en/home/life-science/sequencing/dna-sequencing/targeted-sequencing/targeted-sequencing-ion-torrent-next-generation-sequencing.html.
