title: Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.
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.
authors.affiliation: 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.
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
authors.affiliation: Ontario Institute for Cancer Research, MaRS Centre, 661 University Avenue, Suite 510, Toronto, Ontario M5G 0A3, Canada.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Krzyzanowski
authors.affiliation: Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA.
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Jackson
authors.affiliation: Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Egyud
authors.affiliation: Ontario Institute for Cancer Research, MaRS Centre, 661 University Avenue, Suite 510, Toronto, Ontario M5G 0A3, Canada.
authors.forename: Lincoln
authors.initials: L
authors.lastname: Stein
authors.affiliation: Department of Surgery, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA godfreyt@bu.edu.
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D001706:Biopsy; D002460:Cell Line; D058893:DNA Barcoding, Taxonomic; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers