Ion AmpliSeq™ from Thermo Fisher Scientific is a targeted PCR-based transcriptome profiling technology that allows users to measure the expression levels of over 20,000 genes, even in challenging low-quantity or degraded RNA samples such as from formalin-fixed paraffin-embedded (FFPE) tissue.
But with limited sample multiplexing capabilities, a relatively high cost per sample, and reliance on pre-selected target primer panels, Ion AmpliSeq™ might not be the most appropriate technology for large-scale exploratory transcriptome profiling studies.
In this article, we highlight the workflow and limitations of Ion AmpliSeq™ and suggest recent alternatives.
The Ion AmpliSeq™ technology
The backbone of Ion AmpliSeq™ is the selected pool of oligonucleotide primer pairs designed to amplify specific genomic regions or target mRNA transcripts (Thermo Fisher Scientific 2023a). The technology is reliant on sensitive PCR amplification, so gene expression results can be obtained from as little as 10 ng of total RNA from FFPE samples, cells, or other sources, and is compatible with most species.
The most comprehensive targeted kit currently available for humans is the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit with oligonucleotide designs targeting 20,802 genes that represent >95% of human genes in the RefSeq database (Thermo Fisher Scientific 2023b). However, this pre-designed targeted approach cannot detect novel transcripts, alternative splicing events, or provide reads covering the entire transcriptome. Users do have the option to design custom primers for regions of interest, such as specific splicing events or fusion transcripts, but not on a transcriptome-wide scale.
A glance at the workflow
The workflow begins with the reverse transcription of total RNA with the SuperScript™ VILO™ cDNA synthesis kit. Targets are then amplified by PCR, and primers are partially digested to eliminate primer dimers (Thermo Fisher Scientific 2023c).
If multiple libraries are to be multiplexed on a single chip, each library must then be barcoded with adapters before sequencing. Only approximately 16 samples can be multiplexed per run when using an Ion 550™ chip, possibly hindering studies with large sample sizes (Thermo Fisher Scientific 2023d).
For comparison, recent technologies such as MERCURIUS™ FFPE-seq, MERCURIUS™ DRUG-seq and MERCURIUS™ BRB-seq allow users to multiplex up to 384 samples in the same library pool very early in the workflow, significantly reducing cost, hands-on time and consumable use while boosting sample throughput.
Next, the unamplified libraries must be purified and quality-controlled to ensure each library has the same concentration before library pooling. Clonal amplification of the libraries occurs via emulsion PCR on beads using the Ion Chef™ system, which then loads purified templated beads onto Ion chips for loading into the sequencing machine. Prepared libraries are sequenced on an Ion Proton™ sequencer, such as the Ion S5 XL system that produces 100–130 million sequencing reads from Ion 550™ chips.
As with other approaches, such as the MERCURIUS™ family of technologies from Alithea Genomics, Ion AmpliSeq™ lends itself to automation as liquid handling systems can be used throughout to significantly reduce hands-on time at every stage.
An unnecessary compromise?
Overall, Ion AmpliSeq™ targeted sequencing technology provides researchers with transcriptional read-outs of genes contained in pre-selected panels but misses the mark if unbiased whole transcriptome profiling is required, such as for exploratory studies on large numbers of samples.
The targeted technology is suited to the interrogation of selected available panels of oncogenic, apoptotic, or other biological pathway biomarkers, but thanks to advances in unbiased RNA-sequencing-based technologies, this level of information is now provided at lower cost with no compromise on the number of genes or multiplexed samples included, even for degraded RNA samples as is now possible with MERCURIUS™ FFPE-seq.
Our follow-up article explores how the Ion AmpliSeq™ technology has been used in large-scale studies to advance our understanding of human health and disease. We also compare it to the MERCURIUS™ family of technologies to examine how novel bulk mRNA-seq methods are becoming more appealing than targeted technologies in terms of scalability, cost per sample, and data quantity for large-scale studies.
Please contact us to find out more about how Alithea Genomics can help your next ultra-high-throughput sequencing project.
- Thermo Fisher Scientific (2023a) ‘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
- Thermo Fisher Scientific (2023b) ‘Ion AmpliSeq™ Transcriptome Human Gene Expression Kit’ Available at: https://www.thermofisher.com/order/catalog/product/A26327.
- Thermo Fisher Scientific (2023c) ‘Ion AmpliSeq™ Transcriptome Human Gene Expression Kit - USER GUIDE’. Available at: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0010742_AmpliSeqTranscriptomeHumanGeneExpr_UG.pdf.
- Thermo Fisher Scientific (2023d) ‘Ion 550™ Chip Kit’. Available at: https://www.thermofisher.com/order/catalog/product/A34537.