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Göteborgs universitets publikationer

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

Författare och institution:
Anders Ståhlberg (Sahlgrenska Cancer Center); Paul M Krzyzanowski (-); Jennifer B Jackson (-); Matthew Egyud (-); Lincoln Stein (-); Tony E Godfrey (-)
Publicerad i:
Nucleic acids research, 44 ( 11 ) s. no. e105
ISSN:
1362-4962
Publikationstyp:
Artikel, refereegranskad vetenskaplig
Publiceringsår:
2016
Språk:
engelska
Fulltextlänk:
Sammanfattning (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.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
MEDICIN OCH HÄLSOVETENSKAP ->
Medicinska grundvetenskaper
Postens nummer:
235525
Posten skapad:
2016-04-29 08:32
Posten ändrad:
2016-08-15 08:53

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