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

Artikel i vetenskaplig tidskrift
Författare Anders Ståhlberg
P. M. Krzyzanowski
M. Egyud
Stefan Filges
L. Stein
T. E. Godfrey
Publicerad i Nature Protocols
Volym 12
Nummer/häfte 4
Sidor 664-682
ISSN 1754-2189
Publiceringsår 2017
Publicerad vid Institutionen för biomedicin, avdelningen för patologi
Sahlgrenska Cancer Center
Sidor 664-682
Språk en
Länkar doi.org/10.1038/nprot.2017.006
Ämnesord rare mutations, cancer-patients, plasma, resistance, cytology, carcinoma, diagnosis, therapy, Biochemistry & Molecular Biology
Ämneskategorier Cancer och onkologi, Biokemi och molekylärbiologi

Sammanfattning

\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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