Key Features

Ultrasensitive

Limit of detection down to 0.1% VAF

 

Quantitative

Quantify mutation VAF in original sample with 95% agreement with ddPCR

Customized

Fully-automated customized BDA designs delivered in less than 3 business days

Sample Flexibility

Compatible with several sample types

Comprehensive

Wide range of mutations covered

User-friendly

Easy workflow with customizable assays

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Benefits

  • Detectable Mutations: Applicable to most mutations
  • Compatible with plasma samples, tumor biopsies, paraffin-embedded sections (FFPE), fresh frozen tumors, and liquid biopsies
  • Requires 10ng-1ug DNA (e.g. from 5-10 ml of blood plasma, or 1 curl of 0.1-10um thickness fresh frozen tumor section or 1 curl of 1-10um thickness FFPE)
  • Has an algorithmic design process for BDA enabling delivery of customized NGSure in as little as two business days.

Sample Data

By applying the BDA technology, sequences with rare mutations will be selectively amplified and enriched, enabling low-VAF mutations in samples to be easily identified via qPCR. The results will be confirmed by Sanger sequencing.

Workflow

Step 1: Order your customized Kit/Service online

Simply input the genome coordinates of your locus of interest on NGSure website.

If you are ordering NGSure assay kits to validate your samples by yourself, kits with arithmetically designed BDA sets will be shipped to you.

If you are ordering validation service, please ship your genomic DNA samples to us and we will provide you with the base identity and VAF of the locus of interest in your samples.

Step 2: qPCR BDA Reaction

qPCR BDA reaction using NuProbe’s NGSure kits
2 hrs (30 min hands-on)

Step 3: Sanger Sequencing (Optional)

Sanger sequencing using sequencing primers provided in NuProbe’s NGSure kits.

Sample preparation, including PCR product purification, cycle sequencing and sequencing clean-up.:
3.5 hrs (1hr hands-on)

Sequencing: Purified Sanger products are analyzed by capillary electrophoresis.
2 hrs (15 min hands-on)

Overall turnaround time for STEP 2+STEP 3: 7.5 hrs | Overall hands-on time for STEP 2+STEP 3: 2 hrs
If you are ordering NGSure service, we will perform STEP 2 and 3 for you and provide a report with the base identity and VAF information of the locus of interest in your samples.

Compatible Instruments

qPCR

BioRad CFX96


ABI 7500


Cobas z480

Sanger sequencing

ABI 3500 Dx Genetic Analyzer

Background

For research use only. Not for use in diagnostic procedures.

Next-generation sequencing (NGS) exhibits limited clinical sensitivity for somatic mutations, particularly for tumor tissue and cell-free DNA samples with mutations of low variant allele fractions. Therefore, the American College of Medical Genetics and Genomics recommends clinical laboratories to validate reported NGS variants using an orthogonal experimental method to rule out potential errors.

However, currently available validation assays are either time-consuming or can only cover limited types of mutations. Consequently, clinical laboratories often do not validate NGS results or do not report low frequency mutations, adversely affecting healthcare decisions and patient care.

NGSure is a cost-effective product line of qPCR and Sanger-based assays for the rapid and sensitive validation of variants identified by NGS. The kit uses NuProbe’s PCR-based multiplexed Blocker Displacement Amplification (BDA) technology to enable the selective amplification of low abundant sequence variants in a background of wildtype DNA.

NuProbe has developed NGSure, a new and cost-effective product line for the rapid and accurate orthogonal validation of putative mutations found through NGS. The kit uses NuProbe’s PCR-based multiplexed Blocker Displacement Amplification (BDA) technology to enable the selective amplification of low abundant sequence variants in a background of wildtype DNA. Compatible with standard Sanger sequencing, NGSure detects mutations with 100-fold better analytical sensitivity (0.1% VAF limit of detection), including for algorithmically designed customized assays that can be provided to customers in as few as three (3) business days.