Archer™ FUSIONPlex™ Lung v2 panel

FUSIONPlex assays use RNA due to the biological relevance for fusions, cost efficiency, and faster turnaround time with this input type. 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. Many of the translocations that occur in a cell may not be expressed and thus potentially have little or no biological relevance, so DNA is not the ideal input to interrogate for oncogenic fusions. Since RNA is the intermediate product of gene expression, it’s ideal for detecting fusions. Detecting fusions with DNA requires more sequencing resources with reduced efficiency and potentially lengthening turnaround time.

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Yes, DNA and RNA from a single sample can be analyzed with VARIANTPlex™ and FUSIONPlex™ panels to provide a genomic profile of the cancer. For comprehensive genomic profiling, pair VARIANTPlex Complete Solid Tumor and FUSIONPlex Pan Solid Tumor v2 panels to interrogate 511 genes for SNVs, indels, CNVs, fusions, exon-skipping, and splicing variants, as well as assessment of MSI and TMB. Additionally, the IMMUNOVerse™ TCR panel can provide tumor-infiltrating lymphocyte information relevant for solid tumor characterization. All Archer panels have parallel workflows, allowing for streamlined comprehensive solid tumor profiling.

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Yes, hotspot SNV/indel targets are included in many of our FUSIONPlex panel designs. The hotspot mutations listed in Archer™ product inserts are intentionally targeted by the assay designs. Note that for SNV/indel targets which 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 6.2.8 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, but labs are not prevented from performing SNV/indel calling on Ion Torrent libraries. Users are currently allowed 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 input to interrogate for certain variants. For detection of those variants, VARIANTPlex™ panels can be ran in parallel with FUSIONPlex for comprehensive genomic profiling.

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The opposing primer-based approach requires target-specific forward and reverse primers designed for both binding sites. This means it’s only possible to interrogate fusions that are known at the time of assay development. Therefore, unknown or novel fusions, including those relevant for solid tumor and blood cancer research, will be missed. 

Anchored Multiplex PCR (AMP), on the other hand, amplifies from one gene-specific primer and one universal primer site in the adapter region, which is ligated to all fragments in the starting material. This enables all possible fusions, regardless of the partner, to be detected, ensuring you receive more relevant information from the sample.

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Gene fusions, both known and novel, act as driver mutations in multiple cancer types by causing:

• Deregulation of one of the partner genes. For example, increased expression of an oncogene due to upstream fusion of a strong promoter.
• Formation of an oncogenic fusion protein. For 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. A “novel fusion” describes a transcript in which an unknown partner is fused to a gene that is known to be involved in the specific cancer type. For example, an unknown NTRK3 fusion that leaves the kinase domain intact is likely to be functionally involved in tumor origination or progression.

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