Gene fusions were detected by RNA sequencing using either the ArcherDx fusion assay (Archer FusionPlex Solid Tumor panel) or whole transcriptome sequencing assay (Illumina NovaSeq platform (Illumina, Inc., San Diego, CA). Variants of genes were pre-determined for their cancer-related and clinical significance interpreted by board-certified molecular geneticists and categorized as pathogenic (P), presumed pathogenic (PP), variant of unknown significance (VUS), presumed benign (PB), or benign (B), according to ACMG (American College of Medical Genetics and Genomics) standards.27 (link)
Fusionplex solid tumor panel
Manufactured by Illumina
The FusionPlex Solid Tumor panel is a laboratory equipment product designed for the analysis of genomic alterations in solid tumor samples. It provides a comprehensive targeted sequencing solution for the detection of gene fusions, insertions, deletions, and other relevant mutations.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq platform, by Illumina (3 mentions)
Miseq platform, by Illumina (2 mentions)
Nextseq platform, by Illumina (1 mentions)
Rna ffpe tissue extraction kit, by Agilent Technologies (1 mentions)
Agilent sureselect human all exon v7 bait panel, by Agilent Technologies (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Miseq platform, by Agilent Technologies (1 mentions)
4 protocols using fusionplex solid tumor panel
1
Fusion detection and variant analysis
2
Comprehensive Tumor DNA Sequencing Protocol
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Tumor only targeted DNA sequencing was performed by Caris Life Sciences (Irving, Texas) on genomic DNA isolated from FFPE tumor samples using a minimum of 50 ng of DNA, of which >20% was required to be of tumor origin, as assessed by H&E staining. The DNA was sequenced via the Agilent custom designed SureSelect XT assay (Caris MI TumorSeek 592-Gene NGS Panel, details at www.carislifesciences.com ) on the Illumina NextSeq platform. Copy number variation was determined by comparing the depth of sequencing of genomic loci to a diploid control as well as the known performance of these genomic loci. Gene fusion and variant transcript detection were performed on mRNA isolated from FFPE tumor samples using the Archer FusionPlex Solid Tumor Panel and sequenced on Illumina MiSeq.
Variants were analyzed with maftools40 (link). For visualization, only the most disruptive variant based on SIFT and PolyPhen classifications was chosen when a sample had multiple variants per gene. TCGA variant data was obtained from cBioPortal41 (link).
Variants were analyzed with maftools40 (link). For visualization, only the most disruptive variant based on SIFT and PolyPhen classifications was chosen when a sample had multiple variants per gene. TCGA variant data was obtained from cBioPortal41 (link).
3
Detecting RET Fusion via Targeted RNA Sequencing
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RET fusion was detected by either the ArcherDx fusion assay (Archer FusionPlex Solid Tumor panel) or the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) with the use of the Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). For the ArcherDx fusion assay, formalin-fixed paraffin-embedded tumor samples were microdissected to enrich the sample to ≥20% tumor nuclei, and mRNA was isolated and reverse transcribed into complementary DNA (cDNA). Unidirectional gene-specific primers were used to enrich for target regions, followed by Next-Generation sequencing (Illumina MiSeq platform). For fusion detection using the Illumina NovaSeq platform, FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity was determined using the Agilent TapeStation.
4
Detecting Gene Fusions via NGS Sequencing
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Gene fusions were detected by ArcherDx fusion assay (Archer FusionPlex Solid Tumor panel) on Illumina MiSeq platform or WTS (Agilent SureSelect Human All Exon V7 bait panel) on Illumina NovaSeq platform (Illumina, Inc., San Diego, CA). The formalin-fixed paraffin-embedded tumor samples underwent pathology review and were micro-dissected to enrich the tumor nuclei prior to mRNA extraction. For Archer assay, unidirectional gene-specific primers were used to enrich for target regions, followed by NGS (Illumina MiSeq platform). Targets included 52 genes, and the full list can be found at http://archerdx.com/fusionplex-assays/solid-tumor . For WTS, biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets and the bait-target complexes were amplified in a post capture PCR reaction. The resultant libraries were quantified, normalized and the pooled libraries are denatured, diluted, and sequenced; the reference genome used was GRCh37/hg19.
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