Archived tumor samples, where clinical scientists preserve their biopsies by fixing with formalin and embedding in paraffin (FFPE samples), are a rich source of material for retrospective research studies. However, the process of chemically crosslinking and subsequently extracting nucleic acids from such samples often results in degraded and chemically modified DNA or RNA. These modifications often present barriers for downstream analysis. Nevertheless, by combining nucleic acid extraction kits optimized for FFPE with Sanger sequencing and the Applied Biosystems™ Minor Variant Finder software, we have defined a workflow that allows clinical researchers to obtain variant allele frequencies as low as 5%.
- First we obtained a large number of FFPE tumor samples from a commercial source.
- We extracted DNA from these samples using the Thermo Fisher (Ambion) RecoverAll kit.
- Our DNA yields varied from 2.2µg to 112ng from a single slice.
- We then sequenced these samples using the Ion AmpliSeq™ Oncology Focus panel and identified allelic variants in each sample.
- Then we extracted DNA from independent slices from the same tissue block.
- We verified the allelic variant and frequency of the allele using Thermo Fisher’s Big Dye Direct and Big Dye Exterminator Sanger sequencing kits, analyzed on a Thermo Fisher Scientific 3500 Capillary Electrophoresis instrument, and analyzed the traces using the Minor Variant Finder software.
- We compared the variant frequencies obtained by next generation sequencing and Sanger sequencing (see figure).
- Overall, the correlation between the two systems was excellent.
- The R2 value between these very different sequencing systems was around 0.83.
- Notably, the results correlated well when as little as 1ng of extracted DNA was used in the sequencing reactions
Our results demonstrate that excellent-quality sequencing results can be obtained from archival tumor samples using a complete Thermo Fisher Scientific workflow. This should give scientists and clinical researchers the confidence to utilize their libraries of samples on slides to obtain high-quality genomic data.
Download the application note: Sanger sequencing using Ion AmpliSeq primers and libraries
This application note presents a workflow for extremely limited genomic DNA 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.
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