Top 6 FAQs on RNA fusion analysis

August 24, 2021

Combining RNA fusion analysis, gene expression profiling and SNV calling in a single assay can fast-track your path to comprehensive oncology insights. However, false positives and reduced sensitivity are key bottlenecks with many existing NGS workflows.

Our resident RNA fusion expert, Dr. Song Tian, recently presented a live webinar and Q&A session on how to optimize RNA fusion analysis to unlock multiple biomarker insights.

Explore the answers to the top 6 FAQs compiled from this live webinar session.

What is the benefit of detecting RNA fusions simultaneously with gene expression and CNVs?

NGS provides a powerful tool for biomarker studies. A new trend in NGS is to combine multiple analytes to better understand the biological and pathological changes that distinguish different groups, such as benign or malignant, and response vs. non-response to therapy. Researchers have already started to dabble in this area by using various RNA molecules as biomarkers and adopting a multimodal approach for the diagnosis and prognosis of disease. One example is a recent study by Cani et al. in which an NGS-based urine test was developed for early detection of aggressive prostate cancer. In this study, they found 84 relevant biomarkers, including T2:ERG gene fusion, PCA3 lncRNA, mRNAs and other lncRNAs and expressed mutations. 

How would I know if I needed full exon coverage or partial exon coverage for a novel fusion? Just to be on the safe side, should I use full-exon designs?

If you want to cover a full exon at all locations within this exon, it can be complicated. In most cases, the fusion still follows the standard splicing patterns identified by the base model of the ensemble or the comprehensive model of the rare transcript splicing site. But occasionally, there is a new splicing site, so if you have no information about this novel splicing site and want to cover all the possibilities, you need to define multiple target sites. To input every nucleotide as a target, start at the boundary towards the end of the exon, then we can design the panel based on that to cover the whole exon location. The primer we design is based on that and will cover any breakpoint – whether it’s in the middle, the 5’ or the 3’ side of exon. So this is a relatively complicated design, but if this is your research need for discovery power, we can do that with the QIAseq RNA Fusion XP Custom Panels.

Is QIAseq panel technology just like nested PCR that other companies use?

No, it is totally different. Nested PCR needs two gene-specific primers on one side for enrichment, posing limitations on primer design. That is a challenge when designing a custom panel if you have an unknown target list and need to modify and add additional new targets. With single primer extension, you have the flexibility to add more primers and the flexibility of any sequential location, which is a limitation of nested PCR.

Is there a difference in universal controls used in custom vs. catalog panels?

The controls we add are the same by default. We have universal controls added to each cataloged panel, and we also use these controls automatically for custom panels. In addition, you can also add your own controls. For example, if you have a gene expression target to use as a reference, you can add that. The QIAseq analysis portal will report your target and the standard control target together so you can decide what to use based on your own needs.

I understand that you are using a custom read 1 primer in this product. Is there a specific reason for this?

After we generate the library based on single primer extension technology, we can start the reads from our primer site, which means we know where it is located and where it goes. That way, we are sure that our reads will cover the fusion junctions. This is a unique design and we can only do this with a custom read 1 primer. If you use a regular primer, you may have some extra common regions, and once you reduce the diversity for the initial part of reads, you waste a lot of reads, which means you have less signal that can be used for the fusion call. This is the most important benefit of using this read 1 primer.

I'm interested in a custom design, who should I contact?

If you are interested in designing your own RNA fusion panel, check out the custom design portal on GeneGlobe and follow the simple instructions on the side panel. If you have any questions about your design, please contact our technical support team in the design builder and we would be happy to help.

Find out more about QIAseq Fusion XP Targeted Panels here.
View the on-demand webinar here.

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