Molecular Diagnosis & Therapy

, Volume 22, Issue 5, pp 603–611 | Cite as

Validation Strategy for Ultrasensitive Mutation Detection

  • Marija Debeljak
  • Michael Noë
  • Stacy L. Riel
  • Lisa M. Haley
  • Alexis L. Norris
  • Derek A. Anderson
  • Emily M. Adams
  • Masaya Suenaga
  • Katie F. Beierl
  • Ming-Tseh Lin
  • Michael G. Goggins
  • Christopher D. Gocke
  • James R. EshlemanEmail author
Original Research Article



Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients.


The objective of this study was to develop a method to correct errors in low-level cell line mixes.

Materials and Methods

We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing.


We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99).


The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.



We thank Dr. Zhen Zhang for his insight and expert statistical analysis. We acknowledge Drs. Lori Sokoll, Jun Yu, Maria Bettinotti, Annette Jackson, Bo Song (Bio-Rad), and Kenneth Pienta, in addition to Brian Iglehart and Don Vindivich, for helpful discussions.

Compliance with Ethical Standards

Conflict of interest

Marija Debeljak, Stacy L. Riel, Lisa M. Haley, Alexis L. Norris, Derek A. Anderson, Emily M. Adams, Masaya Suenaga, Katie F. Beierl, Ming-Tseh Lin, Michael G. Goggins, Christopher D. Gocke, James R. Eshleman, and Michael Noë have no conflicts of interest that are directly relevant to the content of this work.


This work was funded in part by The Sol Goldman Pancreatic Cancer Research Center, the Stringer Foundation, the Michael Rolfe Pancreatic Cancer Foundation, Mary Lou Wootton Pancreatic Cancer Research Fund, and the Institute for Clinical and Translational Research (ICTR) Accelerated Translational Incubator Pilot (ATIP) Program.

Ethical Approval and Informed Consent

No patients were enrolled and nor was protected health information used in this study.

Supplementary material

40291_2018_350_MOESM1_ESM.pdf (633 kb)
Supplementary material 1 (PDF 633 kb)


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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Marija Debeljak
    • 1
  • Michael Noë
    • 1
    • 2
  • Stacy L. Riel
    • 1
  • Lisa M. Haley
    • 1
  • Alexis L. Norris
    • 1
  • Derek A. Anderson
    • 1
  • Emily M. Adams
    • 1
  • Masaya Suenaga
    • 1
  • Katie F. Beierl
    • 1
  • Ming-Tseh Lin
    • 1
  • Michael G. Goggins
    • 1
    • 2
    • 3
    • 4
  • Christopher D. Gocke
    • 1
    • 2
  • James R. Eshleman
    • 1
    • 2
    • 4
    Email author
  1. 1.Department of PathologyJohns Hopkins University, Johns Hopkins Medical InstitutionsBaltimoreUSA
  2. 2.Department of OncologyJohns Hopkins University, Johns Hopkins Medical InstitutionsBaltimoreUSA
  3. 3.Department of MedicineJohns Hopkins University, Johns Hopkins Medical InstitutionsBaltimoreUSA
  4. 4.The Sol Goldman Pancreatic Cancer Research CenterJohns Hopkins University School of MedicineBaltimoreUSA

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