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It all comes down to biological relevance, cost and turn-around-time. Translocations can occur anywhere in the genome, including introns and other non-coding sequences. They can also occur within the coding regions of genes with limited expression patterns. What this means is that many of the translocations that occur in a cell may not be expressed and thus have little or no biological relevance. For this reason, DNA is not the ideal substrate to search for oncogenic fusions. RNA, on the other hand, is the intermediate product of gene expression and is ideal for detecting fusions, because you are only looking at those that are expressed and potentially oncogenic.

Searching for translocations in non-coding regions of the genome is time consuming and expensive. For example, DNA-based hybrid capture techniques tile over intronic regions, which can be repetitive, homopolymer-prone and span 100kb or more. This approach requires more probes, more space on your sequencer and more input material. And even then, coverage can be spotty. On the other hand, FusionPlex assays use RNA transcripts and place gene-specific primers near known fusion breakpoints, so you can identify translocations with a single primer.

Since FusionPlex assays combine primers for multiple fusion targets, you can efficiently detect more fusions with less reads and input material. FusionPlex assays use RNA to detect fusions and are better, faster and cheaper than DNA-based hybrid capture techniques. Detect fusions the Archer way, with one of the many FusionPlex assays.

Gene fusions act as driver mutations in multiple cancer types by causing:

  • Deregulation of one of the partner genes (example: increased expression of an oncogene due to upstream fusion of a strong promoter)
  • Formation of an oncogenic fusion protein (example: constitutive activation of a receptor tyrosine kinase due to fusion of a dimerization domain)
  • Inactivation of a tumor suppressor (for example: truncation of a functional domain)

Many known driver fusions involve a targetable partner.

In the context of FusionPlex® “unknown fusion” describes a transcript in which a novel partner is fused to a gene that is known to be involved in the specific cancer type. For instance, an unknown NTRK3 fusion detected in a patient with cancer that leaves the kinase domain intact is likely to be functionally involved in disease origination or progression. It is important to detect this unknown fusion because it may be actionable.

DNA-based NGS approaches rely on hybrid capture technology and require tiling of the intronic regions to capture the exact breakpoint. The problem is that intronic regions can be very large, have highly repetitive sequences and can be homopolymer prone. Since numerous introns have gaps in coverage, like the critical one in ROS1 between exon 32 and 31 there are regions that can map to over 10,000 different spots in the genome making adequate coverage over this region unobtainable.

The intron is over 7kb. Therefore, tiling these intronic regions can get extremely expensive. For example, if we were to take our RNA based, fusion-focused 14 gene lung panel and cover the same fusions with a DNA hybrid capture based approach, it would require a massive amount of probes and sequencing space. Increasing the sequencing cost alone by an order of magnitude.

Additional benefit of using RNA over DNA: non specific mapping as some introns harbor repetitive sequence elements also present elsewhere in the genome.

The majority of our customers validate our panels down to an LOD of 2-3% fusion containing transcript.
Do not pre-treat the RNA. Pre-treatments that should be avoided include:
  • Amplification
  • Shredding or shearing of RNA
  • RT-PCR
  • Staining of FFPE slides
  • Decalcification
Hotspot SNV/InDel targets are included in many of our FusionPlex panel designs. The hotspot mutations listed in our Product Inserts are intentionally targeted by the assay designs. Note: for SNV/InDel targets that do not appear in our Product Inserts, coverage will occasionally be generated by gene specific primers (GSP) designed to cover fusion breakpoints, therefore, we flag all GSP2s with both the “”FUSION”” and “”SNV”” function flags in our GTFs. Version 6.3 and earlier of Archer® Analysis may not support RNA SNV/InDel variant calling at exon junctions depending on the sequence context (SNVs ≤5bp, Indels ≤30bp). RNA SNV/InDel mutation detection is not formally supported on the Ion Torrent™ Sequencing Platform. We do not however, prevent customers from performing SNV/InDel calling on Ion Torrent libraries, and currently allow users to perform targeted variant detection using a targeted mutation file (TMF). Additional considerations:
  • FusionPlex assays can only be used to interrogate variants that are expressed in the sample.
    • Archer Analysis reports expressed allele frequencies.
  • Inactivating mutations in RNA can increase instability of transcripts via nonsense mediated decay.
    • This can lead to a higher rate of false negative calls when using RNA input.
    • DNA is a better molecule to target for certain variants.

Yes, they are compatible. The FusionPlex Pan-Heme and FusionPlex Pan Solid Tumor require 4.5M and 3.5M reads respectively. That being said, we would recommend using at least an Ion Chip™ 530 or higher. The 530 chip is capable of ~15-20M reads, which will allow for ~5 samples, the 540 chip has 60-80M reads, so you can get about 20 samples and the 550 chip (not compatible on the Ion S5™ System you listed below) is 100-130M reads, so you can at least 28 samples.

The 550 chip would only work on the Ion GeneStudio™ S5 Prime System.

We would recommend the 540 chip for the higher throughput, but it all depends on how many samples you plan to run.

Yes, FusionPlex is compatible with this model. One note though, the S5 will not run an Ion Chip™ 550, if you plan for the higher throughput. You would need the S5 Prime for the 550 chip.

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