Seq+FA: Sanger Sequencing Sensitivity with Fragment Analysis

The Sanger Sequencing + Fragment Analysis (Seq+FA) application combines traditional Sanger sequencing with the high-precision fragment sizing capability of an Applied Biosystems® genetic analyzer. By combining the power of the two technologies, this new application:

  • Associates base calling and associated quality values
  • Associates size, peak height, and peak area for each nucleotide
  • Improves sequencing sensitivity to a level of ~5%
  • Enables easy sample comparison

The Seq+FA application is recommended for:

  • Analysis of minor variations like somatic mutations or polyploid genome analysis
  • Assessing the amount of methylation in promoter regions
  • Distinguishing heterozygous insertions from deletions
The sensitivity limit of the Seq+FA application was tested against other major techniques. To read the complete study, download “Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection”.

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Seq+FA workflow. By adding a size standard to a portion of the BigDye® Terminator v1.1 sequencing reaction and separating it using the fragment analysis run module, users can gather data on peak height and ratios and can compare the base profiles of different samples—and then correctly correlate all of that data back to the nucleotide sequence.

Webinars

Watch this short webinar to learn more about the Seq+FA application.

Workflow

Sanger sequencing is performed using BigDye® Terminator version 1.1 chemistry. The purified sequencing reaction is run on the capillary electrophoresis instrument in sequencing mode, generating a “.ab” output file with data for sequence analysis.

This step is optional and can be performed for a reference sample only. This reference sequence will be used to determine the sequence and compare the sequencing profile with the fragment analysis profile.

Learn more about Sanger sequencing

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