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Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection

Qiang Gan, Andrew Fu, Fang Liu, Shuo Shen, Maidar Jamba, Wei Liu, Mike Powell, Aiguo Zhang, View ORCID ProfileMichael Sha
doi: https://doi.org/10.1101/2021.08.14.456349
Qiang Gan
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Andrew Fu
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Fang Liu
2AdvancedSeq, 6654 Owens Drive, Pleasanton, CA 94588
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Shuo Shen
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Maidar Jamba
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Wei Liu
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
2AdvancedSeq, 6654 Owens Drive, Pleasanton, CA 94588
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  • For correspondence: wei.liu@advancedseq.com msha@diacarta.com
Mike Powell
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Aiguo Zhang
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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Michael Sha
1DiaCarta Inc., 4385 Hopyard Rd., Suite 100, Pleasanton, CA 94588
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  • ORCID record for Michael Sha
  • For correspondence: wei.liu@advancedseq.com msha@diacarta.com
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Abstract

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 Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 15, 2021.
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Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection
Qiang Gan, Andrew Fu, Fang Liu, Shuo Shen, Maidar Jamba, Wei Liu, Mike Powell, Aiguo Zhang, Michael Sha
bioRxiv 2021.08.14.456349; doi: https://doi.org/10.1101/2021.08.14.456349
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Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection
Qiang Gan, Andrew Fu, Fang Liu, Shuo Shen, Maidar Jamba, Wei Liu, Mike Powell, Aiguo Zhang, Michael Sha
bioRxiv 2021.08.14.456349; doi: https://doi.org/10.1101/2021.08.14.456349

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