title: Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.
abstract: 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.
authors.affiliation: Department of Pathology and Genetics, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Anders
authors.identifier: 0000-0003-4243-0191
authors.initials: A
authors.lastname: Ståhlberg
authors.affiliation: Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Krzyzanowski
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Egyud
authors.affiliation: Department of Pathology and Genetics, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
authors.forename: Stefan
authors.identifier: 0000-0002-5994-6699
authors.initials: S
authors.lastname: Filges
authors.affiliation: Ontario Institute for Cancer Research, MaRS Centre, Toronto, Ontario, Canada.
authors.forename: Lincoln
authors.initials: L
authors.lastname: Stein
authors.affiliation: Department of Surgery, Boston University School of Medicine, Boston, Massachusetts, USA.
authors.forename: Tony E
authors.identifier: 0000-0002-3283-6983
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers