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.
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