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Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing

Nature Protocols volume 12, pages 664682 (2017) | Download Citation


Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

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This work was supported by the National Institutes of Health (R21CA172999 to T.E.G.), the Swedish Cancer Society (to A.S.), the Swedish Childhood Cancer Foundation (to A.S.), the Sahlgrenska Academy at the University of Gothenburg (to A.S.).

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Author notes

    • Anders Ståhlberg
    •  & Paul M Krzyzanowski

    These authors contributed equally to this work.


  1. Department of Pathology and Genetics, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

    • Anders Ståhlberg
    •  & Stefan Filges
  2. Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada.

    • Paul M Krzyzanowski
    •  & Lincoln Stein
  3. Department of Surgery, Boston University School of Medicine, Boston, Massachusetts, USA.

    • Matthew Egyud
    •  & Tony E Godfrey


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A.S. and T.E.G. conceived and designed the protocol; P.M.K. and L.S. developed the Debarcer software for data analysis; and M.E. and S.F. contributed to protocol development. A.S., P.M.K., M.E., S.F. and T.E.G. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Anders Ståhlberg or Tony E Godfrey.

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