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

Anders Ståhlberg1, Paul M Krzyzanowski2, Jennifer B Jackson3

  • 1Department 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.

Nucleic Acids Research
|April 10, 2016
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Summary

This study introduces a simple, fast method using barcoded adapter primers to enhance DNA library preparation for liquid biopsies. This technique improves the detection of rare cell-free DNA variants, crucial for non-invasive prenatal testing and cancer biomarker discovery.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Cell-free DNA (cfDNA) in liquid biopsies is vital for non-invasive prenatal testing and cancer detection.
  • Detecting extremely low cfDNA fractions requires ultra-sensitive methods.
  • Current next-generation sequencing (NGS) lacks the sensitivity for rare variant detection.

Purpose of the Study:

  • To develop a simple, fast, and highly sensitive method for cfDNA analysis.
  • To enable accurate detection of rare DNA variants in liquid biopsies.
  • To bridge the sensitivity gap between digital PCR and NGS.

Main Methods:

  • Introduction of molecular barcodes into DNA libraries using barcoded adapter primers with hairpin structures.
  • Protection of barcodes during initial PCR to prevent mis-priming.
  • Generation of consensus reads from barcoded molecules to reduce sequencing noise.

Main Results:

  • A simple and fast method for DNA library preparation from 5 ng of DNA.
  • Successful generation of uniform libraries with up to 31-plex.
  • Detection of variant alleles below 0.1% frequency in cfDNA.
  • Reduced background sequencing noise through consensus read generation.

Conclusions:

  • The developed barcoding method significantly enhances cfDNA detection sensitivity.
  • This approach improves the utility of NGS for analyzing rare variants in liquid biopsies.
  • The method offers a powerful tool for non-invasive prenatal testing and cancer biomarker applications.