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Oncomine™ BRCA Research Assay, Manual Library Preparation (Ion Torrent™)

The Oncomine™ BRCA Research Assay consists of two pools of AmpliSeq™ oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on Ion Torrent™ platforms. The assay is designed to provide sensitive and comprehensive sample amplification of all coding regions of the human BRCA1 and BRCA2 genes. This version of the assay is for manual library preparation. For automated library preparation using the Ion Chef™ System, please see Cat. No. A32841.

Features include:

• Validated for use on Ion 318 and Ion 530 chips
• Detects SNVs, InDels, and large exon/gene deletions/duplications
• Compatible with as little as 10 ng input DNA from formalin-fixed, paraffin-embedded (FFPE) tissue or blood samples
• Highly uniform coverage across all coding exons and splice sites for efficient sequencing and accurate analysis
• Optimized primer design and amplification chemistry enable highly specific target enrichment
• Validated detection of somatic variants to 5% and lower
• Simple and fast workflow produces targeted libraries in 3.5 hours typically

The Oncomine BRCA Research Assay is a complete kit facilitating the amplification of the entire exonic region of both BRCA genes from FFPE tissue or blood samples with as little as 10 ng of input DNA. Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on either the Ion PGM™ or Ion S5™ sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatic workflows within Torrent Suite™ and Ion Reporter™ analysis software that utilize optimized variant calling parameters for SNV, InDel, and large exon/gene deletion/duplication detection. Samples can be processed quickly and easily, and variants detected and identified confidently in either germline or somatic DNA analysis pipelines.

BRCA1 and BRCA2 genes are tumor suppressor genes that code for proteins that are vital components of the homologous recombination pathway of DNA damage repair. BRCA mutations resulting in the deficiency of either gene have been shown to result in inefficient activation of this function and are linked to hereditary predisposition to cancer. Errors in the coding sequence of BRCA genes have been detected in germline and somatic mutations and also occur in tumor samples.

Oncomine Lung Cell-Free Total Nucleic Acid Research Assay (Ion Torrent™)

The Oncomine Lung cfTNA Research Assay is part of a complete solution to detect lung tumor-derived cell-free DNA and RNA (cell-free total nucleic acid; cfTNA) isolated from the plasma fraction of whole blood. It provides the reagents for library construction and a single pool of multiplex PCR primers for preparation of amplicon libraries from cfTNA obtained from the plasma fraction of a single 10 mL tube of whole blood.

Liquid biopsies offer several advantages over conventional solid tumor biopsies:
• Less invasive, enabling them to be taken at multiple time points to monitor progression of the cancer
• Lower cost
• Faster turnaround time from sample to results
• Better represent tumor heterogeneity

The Oncomine Lung cfTNA Research Assay enables the analysis of:
• Hotspot genes (SNVs) and short indels: ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 (~168 hotspots covered)
• Gene fusions: ALK, RET, ROS1
• MET exon 14 skipping
• Copy number gene (CNV): MET

These genes have been identified as frequently mutated in non-small cell lung cancer (NSCLC). Through the use of Tag Sequencing technology, low limits of detection (LOD) can be achieved for different variant types*:
• For SNVs/short indels, an LOD of 0.1% can be achieved with sensitivity of ~90% and specificity of >99%
• For fusions & MET exon skipping, an LOD of 1% can be achieved with sensitivity of >90% and specificity of >99%
• For MET CNV target, detection as low as 1.2-fold amplification can be achieved with sensitivity of >90% and specificity of >99%

The entire workflow from isolation of cfTNA using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit to analysis of samples can be accomplished in just two days using the Ion S5 XL sequencing system.

Technology
cfDNA and cfRNA are found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, a tag sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers (figure below). After amplification, the tagged molecules are grouped based on the tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using the Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.

Unlike other technologies with LODs of 1-5%, the Oncomine Lung cfTNA Research Assay has a flexible detection limit down to 0.1% for SNVs or one mutant copy in a background of 1,000 wild-type copies. To achieve 0.1% LOD, 20 ng of input cfDNA is required. Lower amounts of cfTNA can be used, but the %LOD will be higher depending on the input amount.

Advantages:
• Optimized amplicon design for short cfDNA ensures highest possible capture rate
• Tag Sequencing technology minimizes false positives by removing randomly incorporated errors
• Optimized targeted assay design allows highly multiplexed next-generation sequencing (NGS), reducing
sequencing costs per sample
• Efficient 2-day workflow

Analysis of SNVs and short indels can be achieved using Torrent Suite Software 5.2 or higher. In order to analyze SNVs, short indels, fusion, and CNVs, Ion Reporter 5.6 (cloud- or server-based) is required.

Simplicity, speed, and scalability of TagSequencing technology
The Oncomine Lung cfTNA Research Assay enables cancer genetic studies from just 5 ng of input cfTNA for targeted library construction. The Oncomine Lung cfTNA Research Assay is compatible with FFPE samples for possible concordance studies. Total time to targeted libraries is just four hours. Scalability and flexibility are achieved using the Tag Sequencing Barcode Set 1-24 or 25-48 (Cat. Nos. A31830, A31847) for multiplexing barcoded samples on Ion S5 chips.

*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfTNA isolated from normal healthy donors.

Oncomine™ Myeloid Research Assay (Ion Torrent™)

The Ion Torrent Oncomine Myeloid Research Assay consists of three pools of AmpliSeq oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on Ion Torrent platforms. The assay is designed to provide sensitive and comprehensive sample amplification of relevant DNA mutations and fusion transcripts associated with myeloid disorders in a single NGS run. The panel is comprised of 40 key DNA genes and a broad fusion panel of 29 driver genes to cover the most relevant targets in major myeloid disorders: acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML).

This version of the assay is for manual library preparation. For automated library preparation using the Ion Chef System, please see Cat. No. A32841.

Features include:
• Simple, robust workflow that encompasses mutations and fusions in two DNA and one RNA pool in a single NGS run
• Excellent coverage of challenging targets such as CEBPA and FLT3-ITDs
• Multiplex up to four samples on a single Ion 318 chip or twelve on an Ion 530 chip
• Compatible with as little as 10 ng/pool input DNA or RNA per library from blood or bone marrow samples
• Validated detection of somatic variants down to 5% allele frequency
• Simple and fast workflow produces targeted libraries in 3.5 hours typically
• For use on the Ion PGM and Ion S5 systems with manual or automated library preparation
• Compatible with the Oncomine Knowledgebase Reporter for variant annotation

The Oncomine Myeloid Research Assay is a comprehensive, targeted NGS assay designed to assist hematologists in the understanding of myeloid cancer. The assay is a complete kit facilitating the amplification of multiple gene targets from blood or bone marrow samples with as little as 10 ng of input DNA or RNA per pool. Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on either the Ion PGM or Ion S5 sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatics workflows within Torrent Suite and Ion Reporter analysis software that utilize optimized variant-calling parameters for SNV, InDel, and FLT3 large internal tandem duplication (ITD) detection. Variants can be annotated in the Oncomine Knowledgebase Reporter to generate a sample-specific report. Samples can be processed quickly and easily, and variants detected and identified confidently.

Oncomine™ BRCA Research Assay, Chef-Ready Library Preparation (Ion Torrent™)

The Oncomine™ BRCA Research Assay consists of two pools of AmpliSeq™ oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on Ion Torrent™ platforms. The assay is designed to provide sensitive and comprehensive sample amplification of all coding regions of the human BRCA1 and BRCA2 genes. This version of the assay is for automated library preparation using the Ion Chef™ System. For manual library preparation, please see Cat. No. A32840.

Features include:

• Validated for use on Ion 318 and Ion 530 chips
• Detects SNVs, InDels, and large exon/gene deletions/duplications
• Compatible with as little as 10 ng input DNA from formalin-fixed, paraffin-embedded (FFPE) tissue or blood samples
• Highly uniform coverage across all coding exons & splice sites for efficient sequencing and accurate analysis
• Optimized primer design and amplification chemistry enable highly specific target enrichment
• Validated detection of somatic variants to 5% and lower
• Simple and fast workflow produces targeted libraries in 3.5 hours typically

The Oncomine BRCA Research Assay is a complete kit facilitating the amplification of the entire exonic region of both BRCA genes from FFPE tissue or blood samples with as little as 10 ng of input DNA. Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on either the Ion PGM™ or Ion S5™ sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatic workflows within Torrent Suite™ and Ion Reporter™ analysis software that utilize optimized variant calling parameters for SNV, InDel, and large exon/gene deletion/duplication detection. Samples can be processed quickly and easily, and variants detected and identified confidently in either germline or somatic DNA analysis pipelines.

BRCA1 and BRCA2 genes are tumor suppressor genes that code for proteins that are vital components of the homologous recombination pathway of DNA damage repair. BRCA mutations resulting in the deficiency of either gene have been shown to result in inefficient activation of this function and are linked to hereditary predisposition to cancer. Errors in the coding sequence of BRCA genes have been detected in germline and somatic mutations and also occur in tumor samples.

Oncomine™ Breast cfDNA Research Assay v2 (Ion Torrent™)

The Oncomine Breast cfDNA Assay v2 is part of a complete solution to detect breast tumor-derived DNA (ctDNA) in cell-free DNA (cfDNA). It provides the reagents for library construction and a single pool of multiplex PCR primers for preparation of amplicon libraries from cfDNA obtained from the plasma fraction of a single 10 mL tube of whole blood.

Liquid biopsies offer several advantages over conventional solid tumor biopsies:
• Less invasive, enabling them to be taken at multiple time points to monitor progression of the cancer
• Lower cost
• Faster turnaround time from sample to results
• Better represent tumor heterogeneity

The Oncomine Breast cfDNA Assay v2 enables the analysis of:
• Hotspot genes: AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, PIK3CA, SF3B1, TP53 (~152 hotspots)
• Copy number genes (CNVs): CCND1, ERBB2, FGFR1
• Full length genes: TP53 (~80% coverage)

These genes have been identified as frequently mutated in breast cancer. Through the use of Tag Sequencing technology, low limits of detection (LOD) can be achieved for different variant types*:
• For SNVs/short indels, an LOD of 0.1% can be achieved with sensitivity of ~80% and specificity of >99%
• For detection of TP53 mutations, an LOD of 0.5% can be achieved
• For ERBB2 and FGFR1 CNV targets, detection as low as 1.2-fold amplification can be achieved and for CCND1 CNV target, detection as low as 1.4-fold amplification can be achieved with sensitivity of >90% and specificity of >99%

The entire workflow (figure below), from isolation of cfDNA using the MagMAX Cell-Free DNA Isolation Kit (Cat. No. A29319) to analysis of samples, can be accomplished in just two days using the Ion S5 XL sequencing system.

Technology
cfDNA (ctDNA) is found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, a tag sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers (figure below). After amplification, the tagged molecules are grouped based on the amplified tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using the Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.

Unlike other technologies with LODs of 1–5%, the Oncomine Breast cfDNA Assay v2 has a flexible detection limit of 0.1% for SNVs or one mutant copy in a background of 1,000 wild-type copies. To achieve 0.1% LOD, 20 ng of input cfDNA is required. Lower amounts of cfDNA can be used, but the %LOD will be higher depending on the input amount (figure below).

Advantages:
• Optimized amplicon design for short cfDNA ensures highest possible capture rate
• Optimized targeted assay design allows highly multiplexed next-generation sequencing (NGS), reducing sequencing costs per sample
• Efficient workflow in just two days

Analysis of SNVs and short indels can be achieved using Torrent Suite Software 5.2 or higher. In order to analyze SNVs, short indels, and CNVs, Ion Reporter 5.6 (cloud- or server-based) is required.

Simplicity, speed, and scalability of Tag Sequencing technology
The Oncomine Breast cfDNA Assay v2 enables cancer genetic studies from just 1 ng of input cfDNA for targeted library construction. The cfDNA Assay uses standard PCR equipment and two simple PCR reactions, one to attach the unique molecular tags and the second to amplify the library, for high multiplex PCR-based target selection. Additionally, the Oncomine Breast cfDNA Assay v2 is compatible with FFPE samples for possible concordance studies. Total time to targeted libraries is just 3.5 hours. Scalability and flexibility are achieved using the Tag Sequencing Barcode Sets 1-24 (Cat. No. A31830) or 25-48 (Cat. No. A31847) for multiplexing barcoded samples on Ion S5 chips.

*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfDNA isolated from normal healthy donors.

Oncomine™ Myeloid Research Assay—Chef Ready (Ion Torrent™)

The Ion Torrent Oncomine Myeloid Research Assay consists of three pools of AmpliSeq oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on Ion Torrent platforms. The assay is designed to provide sensitive and comprehensive sample amplification of relevant DNA mutations and fusion transcripts associated with myeloid disorders in a single NGS run. The panel is comprised of 40 key DNA genes and a broad fusion panel of 29 driver genes to cover the most relevant targets in major myeloid disorders: acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML).

This version of the assay is for automated Ion Torrent Chef library preparation. For manual library preparation, please see Cat. No. A32840.

Features include:
• Simple, robust workflow that encompasses mutations and fusions in two DNA and one RNA pool in a single NGS run
• Excellent coverage of challenging targets such as CEBPA and FLT3-ITDs
• Multiplex up to four samples on a single Ion 318 chip or twelve on an Ion 530 chip
• Compatible with as little as 10 ng/pool input DNA or RNA per library from blood or bone marrow samples
• Validated detection of somatic variants down to 5% allele frequency
• Simple and fast workflow produces targeted libraries in 3.5 hours typically
• For use on the Ion PGM and Ion S5 systems with manual or automated library preparation
• Compatible with the Oncomine Knowledgebase Reporter for variant annotation

The Oncomine Myeloid Research Assay is a comprehensive, targeted NGS assay designed to assist hematologists in the understanding of myeloid cancer. The assay is a complete kit facilitating the amplification of multiple gene targets from blood or bone marrow samples with as little as 10 ng of input DNA or RNA per pool. Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on either the Ion PGM or Ion S5 sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatics workflows within Torrent Suite and Ion Reporter analysis software that utilize optimized variant-calling parameters for SNV, InDel, and FLT3 large internal tandem duplication (ITD) detection. Variants can be annotated in the Oncomine Knowledgebase Reporter to generate a sample-specific report. Samples can be processed quickly and easily, and variants detected and identified confidently.

Oncomine™ Immune Response Research Assay (Ion Torrent™)

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 manual library construction 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 manual workflow features high sample multiplexing on Ion S5™ and Ion PGM™ sequencing systems. The entire workflow, from sample extraction to results, can be completed in less than 48 hours. Note: An alternate assay is available (Cat. No. A32928) for the preparation of Ion AmpliSeq libraries in automated mode through 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 run 8 samples per single run
• Efficient workflow that offers sample-to-results in less than 48 hours
• 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)

Oncomine™ Comprehensive Assay v3M (Ion Torrent™)

The Oncomine Comprehensive Assay v3 is a targeted, next-generation sequencing (NGS) assay that enables the detection of relevant SNVs, CNVs, gene fusions, and indels from 161 unique genes to help inform drug discovery research and clinical trial research programs. It provides the reagents for library construction and four pools—two DNA and two RNA—of multiplex PCR primers for preparation of amplicon libraries from formalin-fixed paraffin embedded (FFPE) tumor samples. Designed to help you go from hundreds down to a few relevant cancer drivers, this assay is part of a comprehensive workflow that enables NGS data analysis from as little as 10 ng of nucleic acid per pool. The assay is optimized for sequencing on the Ion S5 System with the Ion 540 Chip to enable up to 8 samples (7 samples and one no-template control) per run.

When combined with Oncomine Knowledge Reporter Software, you can create reports that align labels, guidelines, and clinical trials to your results so you can focus on key drivers of cancer in your research.

Key features of the assay are:
• Enables analysis of variants across 161 genes
• Detection of SNVs, CNVs, gene fusions, and indels
• Robust performance from as little as 10 ng per pool (40 ng total) isolated from FFPE samples including fine needle biopsies
• Characterized with molecular standards and controls
• Content driven by the Oncomine Knowledgebase and experienced scientists helps assure coverage of key targets aligned to published evidence

From sample to answer
The Oncomine Comprehensive Assay is part of an NGS workflow that helps researchers to filter variant results from hundreds to just a few key cancer driver variants. Enabled by Oncomine informatics, this filtering, as well as the annotations from on-market labels, established guidelines (US-NCCN, US-FDA, EMSO, EMA), and global clinical trials, helps you contextualize and interpret findings to move toward the answers you need.

The workflow has been adopted by large-scale national clinical trial research programs including NCI-MATCH (United States) and LC-SCRUM (Japan), and includes:
• Proven Ion AmpliSeq chemistry
• Automated library and template prep on the Ion Chef System
• Scalable sequencing on the Ion S5 Sytem
• Optimized Oncomine informatics

Multiple configurations
The Oncomine Comprehensive Assay v3 comes in the following configurations to match your laboratory needs:

Manual library preparation configuration:
Oncomine Comprehensive Library Assay v3M
This configuration contains all the reagents needed to prepare libraries for 24 or 96 samples:
    • Oncomine Comprehensive Panel v3M DNA—contains the DNA primer pools
    • Oncomine Comprehensive Panel v3M RNA—contains the RNA primer pools
    • Ampliseq Library Plus reagents—allow you to manually create your library
Ion Chef automated library preparation configuration:
Oncomine Comprehensive Library Assay v3C
This configuration contains all the reagents needed to prepare libraries for 32 samples:
    • Oncomine Comprehensive Panel v3C DNA—contains the DNA primer pools
    • Oncomine Comprehensive Panel v3C RNA—contains the RNA primer pools
    • Ion AmpliSeq Kit for Chef DL8 reagents—allows you to automate library prep on the Ion Chef System


Oncomine™ Childhood Cancer Research Assay, Chef-Ready (Ion Torrent™)

The Ion Torrent Oncomine Childhood Cancer Research Assay consists of four pools of Ion AmpliSeq oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on the Ion GeneStudio S5 platforms. The assay is designed to provide researchers with sensitive and comprehensive sample amplification of relevant DNA mutations and fusion transcripts associated with childhood and young adult cancers in a single NGS run. The panel is comprised of targets from 202 unique genes: 130 key DNA genes, 28 copy number variant targets, an expansive fusion panel of 90 driver genes with multiple partners, and 9 expression genes and controls. These cover the most relevant targets in the vast majority of all childhood and young adult oncology research samples.

This version of the assay is for automated library preparation using the Ion Chef System. For manual library preparation, see Cat. No. A36485.

Features include:
• Simple, robust workflow that encompasses mutations and fusions in two DNA and two RNA pools in a single NGS run
• Excellent coverage of all relevant targets
• Multiplex up to eight samples on a single Ion 540 Chip
• Compatible with as little as 10 ng/pool input DNA or RNA per library from blood, bone marrow or FFPE samples
• Validated detection of somatic variants down to 5% allele frequency
• Simple and fast workflow produces targeted libraries in 3.5 hours typically
• For use on the Ion GeneStudio S5 Systems with manual or automated library preparation

The Oncomine Childhood Cancer Research Assay is a comprehensive, targeted NGS assay designed to assist researchers in the understanding of childhood and young adult cancer. The assay is a complete kit facilitating the amplification of multiple gene targets from FFPE, blood, or bone marrow samples with as little as 10 ng of input DNA or RNA per pool.

Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on the Ion GeneStudio S5 sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatics workflows within Torrent Suite and Ion Reporter analysis software that utilize optimized variant calling parameters for SNV, InDel, copy number variant, and fusion transcript detection. Samples can be processed quickly and easily, and variants detected and identified confidently.

Oncomine™ Immune Response Research Assay - Automated (Ion Torrent™)

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)

Oncomine™ Childhood Cancer Research Assay (Ion Torrent™)

The Ion Torrent Oncomine Childhood Cancer Research Assay consists of four pools of Ion AmpliSeq oligonucleotide primers and associated reagents to generate amplicon libraries for next-generation sequencing (NGS) on the Ion GeneStudio S5 platforms. The assay is designed to provide researchers with sensitive and comprehensive sample amplification of relevant DNA mutations and fusion transcripts associated with childhood and young adult cancers in a single NGS run. The panel is comprised of 202 unique genes: 130 key DNA genes, 28 copy number variant targets, an expansive fusion panel of 90 driver genes with multiple partners, and 9 expression genes and controls. These cover the most relevant targets in the vast majority of all childhood and young adult oncology research samples.

This version of the assay is for manual library preparation. For automated library preparation using the Ion Chef System, see the Oncomine Childhood Cancer Research Assay, Chef-Ready.

Features include:
• Simple, robust workflow that encompasses mutations and fusions in two DNA and two RNA pools in a single NGS run
• Excellent coverage of all relevant targets
• Multiplex up to eight samples on a single Ion 540 Chip
• Compatible with as little as 10 ng/pool input DNA or RNA per library from blood, bone marrow, or FFPE samples
• Validated detection of somatic variants down to 5% allele frequency
• Simple and fast workflow produces targeted libraries in 3.5 hours typically
• For use on the Ion GeneStudio S5 Systems with manual or automated library preparation

The Oncomine Childhood Cancer Research Assay is a comprehensive, targeted NGS assay designed to assist researchers in the understanding of childhood and young adult cancer. The assay is a complete kit facilitating the amplification of multiple gene targets from FFPE, blood, or bone marrow samples with as little as 10 ng of input DNA or RNA per pool.

Leveraging the power of Ion AmpliSeq technology, this highly multiplexed NGS assay enables the generation of results from multiple samples in a single run. Designed for use on the Ion GeneStudio S5 sequencing systems, results are delivered in days rather than weeks. The assay is aligned with bioinformatics workflows within Torrent Suite and Ion Reporter analysis software that utilize optimized variant calling parameters for SNV, InDel, copy number variant, and fusion transcript detection. Samples can be processed quickly and easily, and variants detected and identified confidently.