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

Journal article
Authors Anders Ståhlberg
P. M. Krzyzanowski
M. Egyud
Stefan Filges
L. Stein
T. E. Godfrey
Published in Nature Protocols
Volume 12
Issue 4
Pages 664-682
ISSN 1754-2189
Publication year 2017
Published at Institute of Biomedicine, Department of Pathology
Sahlgrenska Cancer Center
Pages 664-682
Language en
Keywords rare mutations, cancer-patients, plasma, resistance, cytology, carcinoma, diagnosis, therapy, Biochemistry & Molecular Biology
Subject categories Cancer and Oncology, Biochemistry and Molecular Biology


\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 (similar to 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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