Sanger Sequencing Using Ion Ampliseq Primers and Libraries
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, we offer 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.
Detailed information about the workflow can be found in the above application note. Highlights include:
- 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 Sanger 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
FFPE DNA sequencing workflow
This application note presents a workflow for extremely limited gDNA samples that uses amplification material from Ion AmpliSeq library preparation as a reservoir for reflex testing of individual targets by Sanger sequencing. These data demonstrate that researchers needing a fast and economical solution for confirmation of uncertain NGS results can rely on the robustness and sensitivity of PCR coupled with Sanger sequencing.