Ion Torrent™

Oncomine™ Breast cfDNA Research Assay v2

Catalog number:  A35865

 Related applications:

Ion Torrent™ Next-Generation Sequencing

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Description

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.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Product Line: Oncomine™
Product Size: 8 reactions
Shipping Condition: Dry Ice

Contents & storage

• 16 µL breast cfTNA panel
• 320 µL cfDNA library PCR master mix
• 832 µL low TE buffer
• 8 µL cfDNA library primer P1
• 8 µL cfDNA library primer A/BC1

Documents

Manuals & protocols