While Sanger sequencing once was considered the gold standard, Dr. Sanger sequencing is no longer the gold standard, he said, because there is potential for allele dropout due to polymorphic positions under primers or unknown heterozygous deletions.
Deignan noted there are cases where a Sanger test is negative while clinical exome sequencing is positive for a particular variant. Another approach is to use Sanger sequencing for confirmation, but Dr. Run dilutions for limit of detection. It is important to correlate sequencing results with tumor input, he said. Copy number alteration can be detected, but there are limitations.
Ultimately, analytical validation of cancer panels should include normal samples, samples with a wide range of mutations, and samples with low mutant allele frequency, Dr. When assessing a new panel, Dr. Additional reference materials for sequencing are under development. Increasingly, the laboratory is cross-validating from one panel to another using tumor samples with mutations at low mutant allele frequency.
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