Confirming Sequencing Results: When Is Orthogonal Testing Needed

Why Confirmation Exists

Sequencing artefacts arise from PCR errors, sequencing chemistry, alignment problems in repetitive or GC-rich regions, and formalin-induced DNA damage that mimics real mutations. Confirmation by an orthogonal method that has different failure modes reduces the false-positive rate.

Sanger sequencing was the traditional confirmatory method; today laboratories may also use a second next-generation sequencing assay, digital PCR or allele-specific PCR.

Which Calls Are Confirmed

High-confidence calls, well-covered positions with a high variant allele fraction and strong read support, are increasingly reported without separate confirmation, supported by robust validation data.

Calls that are more likely to be artefacts are prioritised for confirmation: low variant allele fraction, low coverage, homopolymer or repeat regions, known artefact-prone positions, and any result that will directly drive a major treatment decision.

The Limits of Sanger

Sanger sequencing has a detection limit of roughly 15 to 20 percent variant allele fraction, so it cannot confirm low-level calls that next-generation sequencing can make. For those, a more sensitive orthogonal method is needed.

A failure to confirm a low-level call by Sanger therefore does not necessarily mean the call was wrong.

What Confirmation Does Not Prove

Confirmation shows the variant is really present in the DNA analysed. It does not establish that the variant is somatic rather than germline, that it is clonal, or that it is clinically meaningful.

Those questions require paired normal testing, clonality assessment and interpretation against the evidence base.

Interpretation Notes

If a report notes that a key call was orthogonally confirmed, that adds confidence in the call's technical accuracy.

For low variant allele fraction calls, ask what confirmation method was used, since Sanger cannot resolve them.

Key Takeaways

• ·Orthogonal confirmation guards against sequencing artefacts that mimic real mutations.
• ·Low-VAF, low-coverage and repeat-region calls are prioritised for confirmation.
• ·Sanger sequencing cannot confirm variants below roughly 15 to 20 percent VAF.
• ·Confirmation proves presence, not somatic origin, clonality or clinical meaning.