 Solution

The repetitive tract can cause DNA polymerase slippage during PCR amplification or the sequencing extension itself, generating stutter products. A heterozygous length variant creates overlapping Sanger sequencing traces downstream of the indel because the two templates lose register; this does not by itself indicate random sequencing failure. Tumor heterogeneity or contamination by normal tissue can add further mixed templates. Avoid creating or obscuring the mutation during amplification and avoid interpreting a mixed trace as one clean allele. Use a suitable high-fidelity DNA polymerase, independent amplifications and bidirectional reads, and resolve individual alleles by molecular cloning or an appropriate single-molecule/amplicon sequencing method if necessary. Check for non-specific products and allelic dropout caused by variation in a primer-binding site.