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.

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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.