PT  - JOURNAL ARTICLE
AU  - Qiang Gan
AU  - Andrew Fu
AU  - Fang Liu
AU  - Shuo Shen
AU  - Maidar Jamba
AU  - Wei Liu
AU  - Mike Powell
AU  - Aiguo Zhang
AU  - Michael Sha
TI  - Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection
AID  - 10.1101/2021.08.14.456349
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.08.14.456349
4099  - http://biorxiv.org/content/early/2021/08/15/2021.08.14.456349.short
4100  - http://biorxiv.org/content/early/2021/08/15/2021.08.14.456349.full
AB  - BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.Competing Interest StatementThe authors have declared no competing interest.