Low-Level Somatic Variant Detection in Tumor FFPE Samples by Sanger Sequencing

Molecular profiling of cancers is becoming more and more important in the research area of personalized cancer therapy, or precision oncology. Next-generation sequencing (NGS) provides a valuable method for high-throughput applications when many targets and samples need to be multiplexed and screened. However, for very focused applications such as single-analyte or single-gene targets, there is a clear need for fast, simple, and affordable methods for detecting low-level somatic variants. The improved sensitivity achieved using Applied Biosystems™ Minor Variant Finder Software makes Sanger sequencing an ideal technology for filling this gap.

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Details of this application of Sanger sequencing can be found in the application note featured below, which demonstrates:

  • The power of high-sensitivity Sanger sequencing in one of its ideal applications: oncology research
  • The detection of low-level (down to 5%) somatic variants using Sanger sequencing along with Minor
    Variant Finder Software
  • The utility of Sanger sequencing for first-line screening in addition to its role as the gold-standard confirmatory
    method for NGS
  • The successful interrogation of minimal amounts (as low as 0.1 ng) of DNA from formalin-fixed,
    paraffin-embedded (FFPE) samples
  • The advantages of Sanger sequencing, including simple workflow and data analysis, low cost per sample (at a limited number of targets), and fast turnaround time

A simple and fast workflow for genetic analysis of tumors using Sanger sequencing


1 Isolate DNA
identify variants
2 Perform PCR
choose primers
3 Prepare
prepare

Prepare for sequencing using BigDye Direct Cycle Sequencing Kit

4 Run sequencing
run sequence

Run sequencing using 3500xL Genetic Analyzer

5 Analyze data
analyze data

Analyze data using Minor Variant Finder Software

Application note: Low-level somatic variant detection in tumor FFPE samples by Sanger sequencing

This application note demonstrates that Sanger sequencing with Minor Variant Finder Software is not only an ideal tool for confirmation of minor variants detected by NGS, but also an attractive first-line screening choice when working with a limited number of targets. The workflow presented here can be used to detect a minor variant present at a level as low as 5% in an FFPE sample using ≤1 ng DNA per reaction. This robust and simple Sanger sequencing approach also offers fast turnaround time (~4 hours including data analysis) at a low cost per sample, providing a valuable tool for oncology researchers.