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Sci Rep. 2018 May 9;8(1):7375. doi: 10.1038/s41598-018-25810-0.

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Author information

1
Center for Noninvasive Diagnostics, Translational Genomics Research Institute, Phoenix, AZ, USA.
2
Integrated Cancer Genomics Division & Collaborative Sequencing Center, Translational Genomics Research Institute, Phoenix, AZ, USA.
3
Mayo Clinic Center for Individualized Medicine, Scottsdale, AZ, USA.
4
Department of Oncology, University of Oxford, Oxford, United Kingdom.
5
North Wales Cancer Treatment Centre, Rhyl, United Kingdom.
6
Department of Cancer Biology, Mayo Clinic Arizona, Scottsdale, AZ, USA.
7
Department of Medicine, Division of Hematology/Oncology, University of California Los Angeles Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA.
8
Department of Internal Medicine, Yale University, New Haven, CT, USA.
9
Center for Noninvasive Diagnostics, Translational Genomics Research Institute, Phoenix, AZ, USA. mmurtaza@tgen.org.
10
Mayo Clinic Center for Individualized Medicine, Scottsdale, AZ, USA. mmurtaza@tgen.org.

Abstract

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.

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