The Oncomine™ Immune Response Research Assay is a targeted gene expression assay designed for the Ion™ next-generation sequencing (NGS) platform. This pan-cancer gene expression assay is designed to interrogate the tumor microenvironment to enable mechanistic studies and identification of predictive biomarkers for immunotherapy in retrospective or prospective clinical trial research cohorts. The assay is optimized to measure the expression of genes involved in tumor-immune interactions, including the low-expressing genes involved in inflammatory signaling.

This assay contains the reagents for automated library construction using the Ion Chef™ System and a single pool of primers used to perform multiplex PCR for preparation of amplicon libraries from formalin-fixed paraffin-embedded (FFPE) samples. The assay enables quantitative evaluation of the expression of markers associated with different leukocyte subsets, antigen presentation, checkpoint pathways, and tumor progression. This 400-gene assay is supported with a user-friendly informatics workflow and demonstrates high sensitivity for low-expressing transcripts derived from FFPE samples (request a gene list). The Oncomine Immune Response Research Assay leverages Ion AmpliSeq™ technology to deliver results from as little as 10 ng of total RNA, enabling robust performance from challenging FFPE samples.

Assay workflow
The Oncomine Immune Response Research Assay automated workflow features high sample multiplexing on Ion S5™ sequencing systems. The entire workflow, from sample extraction to results, can be completed in less than 48 hours with less than 45 minutes of hands-on time from DNA to data, and only two pipetting steps per sample. Note: An alternate assay is available (Cat. No. A32881) for the preparation of Ion AmpliSeq libraries in manual mode without use of an Ion Chef System.

Assay performance
• High repeatability and sensitive detection of gene expression across different types of solid tumors
• Low sample input requirement (10 ng of total RNA material)
• High sensitivity detection of low-expressing signaling molecules such as interleukins and interferon gamma
• High sample multiplexing with flexibility to sequence 4, 8, or 32 samples per single run
• Automated workflow that offers sample-to-results in less than 48 hours, with less than 45 minutes hands-on time and only two pipetting steps
• Automated and seamlessly integrated analysis solution
• Interpretation of immune surveillance and tumor progression mechanism with functional annotation of genes and pathways

Informatics
The Oncomine Immune Response Research Assay comes with optimized informatics and visualization software that minimizes the complexity of data analysis. The analysis workflow consists of a data exploration component automated by the Immune Response Torrent Suite™ Plug-in and a two-group differential analysis provided by the Affymetrix™ Transcriptome Analysis Console (TAC). Together these tools offer a streamlined process for common gene expression analyses and exportable data files that are amenable to further exploration with other third-party software. Specifically, these tools provide:
• QC metrics and expression of housekeeping genes to qualify data
• Hierarchical clustering of samples with user-defined gene sets or by all genes on the panel
• Visualization with PCA, heatmaps, and gene expression distribution plots
• Sample correlation using rich correlation plots
• Fold-change estimates in easy-to-use tabular data format (TAC)
• Volcano plots to visualize differentially genes (TAC)