Formalin-fixed paraffin-embedded (FFPE) tissue is a standard sample type in histology and pathology laboratories; however, the fixation process often damages DNA, resulting in a limited amount of starting material for molecular genetic analysis. In order to extract maximal information from minimal sample amounts, we developed a workflow that enables robust genotyping results from less than 1 ng of FFPE DNA. Utilizing Ion AmpliSeq technology with Sanger sequencing, the workflow offers an ideal solution for routine labs working with a limited number of samples/targets, as well as high-throughput labs that need an orthogonal method for confirming minor alleles.
Researchers who need a fast and economical solution for confirmation of uncertain NGS results can rely on the robustness and sensitivity of PCR coupled with Sanger sequencing.
- Sanger sequencing results can be obtained using Ion AmpliSeq library primer sequences or from existing Ion AmpliSeq library pools
- Robust genotyping results using both Ion AmpliSeq next-generation sequencing (NGS) and confirmatory sequencing can be generated from less than 1 ng of FFPE DNA
- Previously sequenced NGS libraries can be used as a direct input for confirmatory Sanger sequencing
Application note: Sanger sequencing using Ion Ampliseq primers and libraries
This study demonstrates how Ion AmpliSeq panels analyzed by Sanger sequencing can be used to verify the presence of unusual or low-frequency alleles in a sample.
Sanger sequencing workflow
Our simple and fast Sanger sequencing workflow can be completed in less than one workday, from sample to answer. We offer Applied Biosystems products to support many steps of our recommended workflow, from PCR amplification to data analysis. Learn more about each step of our recommended Sanger sequencing workflow.
For Research Use Only. Not for use in diagnostic procedures.