Duplex Sequencing

Duplex-Seq is a tag-based, error-correction method to improve sequencing accuracy (Schmitt et al., 2012). In this method, adapters (with primer sequences and random 12 bp indexes) are ligated onto the template and amplified using PCR. Deep sequencing provides consensus sequence information from every unique molecular tag. Based on the molecular tags and sequencing primers, duplex sequences are aligned, determining the true sequence on each DNA strand. The method is estimated to be > 10,000-fold more accurate than conventional NGS (Ahn et al., 2015). A targeted version of duplex sequencing includes 2 rounds of capture to yield read depth as high as 1,000,000x (Schmitt et al., 2015)


  • Can detect a single point mutation among > 107 sequenced nucleotides(Schmitt et al., 2012), (Schmitt et al., 2015)
  • Very low error rate due to duplex tagging
  • PCR amplification errors can be detected and removed from analysis
  • No additional library preparation steps after addition of adapters



Illumina Library prep and Array Kit Selector


Gregory M. T., Bertout J. A., Ericson N. G., Taylor S. D., Mukherjee R., et al. Targeted single molecule mutation detection with massively parallel sequencing. Nucleic Acids Res. 2016;44:e22

Do H. and Dobrovic A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem. 2015;61:64-71

Maslov A. Y., Quispe-Tintaya W., Gorbacheva T., White R. R. and Vijg J. High-throughput sequencing in mutation detection: A new generation of genotoxicity tests? Mutat Res. 2015;776:136-143


Krimmel J. D., Schmitt M. W., Harrell M. I., et al. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues. Proc Natl Acad Sci U S A. 2016;113:6005-6010

Ahn E. H., Hirohata K., Kohrn B. F., Fox E. J., Chang C. C. and Loeb L. A. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing. PLoS One. 2015;10:e0136216

Schmitt M. W., Fox E. J., Prindle M. J., et al. Sequencing small genomic targets with high efficiency and extreme accuracy. Nat Methods. 2015;12:423-425