Ion Torrent™

Oncomine Lung Cell-Free Total Nucleic Acid Research Assay

Catalog number:  A35864

Related applications:

Ion Torrent™ Next-Generation Sequencing

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Description

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.

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

Specifications

Shipping Condition: Dry Ice
Product Line: Oncomine™

Contents & storage

• 16 µL lung 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
• 22 µL SuperScript VILO Master Mix