Sanger Sequencing With 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.

Download app note

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

1 Isolate FFPE DNA
identify variants

Start with 1 ng of FFPE DNA

2 Amplify
choose primers

Amplify with an Ion AmpliSeq library pool or carry out NGS using an Ion AmpliSeq panel

3 Design primers
choose primers

Use Primer Designer tool to add universal M-13 tails to Ion AmpliSeq primers

4 Sequence
run sequence

Conduct Sanger sequencing using the 3500 Genetic Analyzer

5 Analyze data
analyze data

Analyze data using an NGC module or Minor Variant Finder Software

Application note: Sanger sequencing using Ion AmpliSeq primers and libraries

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.

NGS confirmation with Sanger sequencing

Learn about the Sanger sequencing workflow

Sanger Analysis Modules—free cloud-based tools