Sanger 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
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.
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.
The GeneScan™ 600 LIZ® size standard is added to the sequencing reaction and run on the capillary electrophoresis instrument under fragment analysis run conditions, generating a “.fsa” file that allows data analysis with GeneMapper® Software.
Secondary analysis of the sequencing reaction through the fragment analysis workflow allows researchers to access even more data from the same sample. For example, by performing GeneMapper® software analysis on the fragment run, users can calculate peak height and peak ratios and can compare peaks for each base or perform a side-by-side comparison of the single-base profiles from different samples. Sequence data is not gathered in the fragment analysis run, so the combination of these two analyses—the sequencing run and GeneMapper® software analysis of the fragment analysis run—makes it possible to position the fragment analysis data correctly within the nucleotide sequence.