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Nucleic Acids Res. 2016 Jun 20;44(11):e105. doi: 10.1093/nar/gkw224. Epub 2016 Apr 7.

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

Author information

1
Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA Department of Pathology, Sahlgrenska Cancer Center, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 405 30 Gothenberg, Sweden anders.stahlberg@gu.se.
2
Ontario Institute for Cancer Research, MaRS Centre, 661 University Avenue, Suite 510, Toronto, Ontario M5G 0A3, Canada.
3
Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA.
4
Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA godfreyt@bu.edu.

Abstract

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants.

PMID:
27060140
PMCID:
PMC4914102
DOI:
10.1093/nar/gkw224
[Indexed for MEDLINE]
Free PMC Article
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