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Evaluation of pre-analytical factors affecting plasma DNA analysis

Havell Markus, Tania Contente-Cuomo, Winnie S Liang, Mitesh J Borad, Shivan Sivakumar, Simon Gollins, Nhan L Tran, Harshil D Dhruv, Michael E Berens, Alan Bryce, Aleksandar Sekulic, Antoni Ribas, Jeffrey M Trent, Patricia M LoRusso, View ORCID ProfileMuhammed Murtaza
doi: https://doi.org/10.1101/126839
Havell Markus
Translational Genomics Research Institute;
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Tania Contente-Cuomo
Translational Genomics Research Institute;
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Winnie S Liang
Translational Genomics Research Institute;
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Mitesh J Borad
Mayo Clinic;
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Shivan Sivakumar
University of Oxford;
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Simon Gollins
North Wales Cancer Treatment Centre;
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Nhan L Tran
Mayo Clinic;
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Harshil D Dhruv
Translational Genomics Research Institute;
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Michael E Berens
Translational Genomics Research Institute;
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Alan Bryce
Mayo Clinic;
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Aleksandar Sekulic
Mayo Clinic;
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Antoni Ribas
University of California Los Angeles Jonsson Comprehensive Cancer Center;
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Jeffrey M Trent
Translational Genomics Research Institute;
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Patricia M LoRusso
Yale University;
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Muhammed Murtaza
Translational Genomics Research Institute and Mayo Clinic
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  • ORCID record for Muhammed Murtaza
  • For correspondence: mmurtaza@tgen.org
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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 (mean amplicon size: 71 bp and 471 bp respectively). Using this assay, we compared performance of 7 cfDNA extraction kits and found cfDNA yield and fragment size varies significantly between them. We also compared 3 blood collection protocols used to collect plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA BCT tubes at ambient temperature processed within 24 hours and 72 hours of collection). To assess whether cell-stabilizing preservative in BCT tubes introduced noise in cfDNA, we performed digital targeted sequencing. We found no significant differences in cfDNA yield, fragment size and background sequencing noise between these protocols. In 219 clinical samples tested for quality using the ddPCR assay, cfDNA fragment size was significantly shorter in plasma samples immediately processed for ctDNA analysis compared to archived samples, suggesting background DNA contributed by lysed peripheral blood cells. In summary, we describe a multiplexed ddPCR approach that enables cfDNA quality assessment and could inform the design of future circulating tumor DNA studies.

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Posted April 18, 2017.
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Evaluation of pre-analytical factors affecting plasma DNA analysis
Havell Markus, Tania Contente-Cuomo, Winnie S Liang, Mitesh J Borad, Shivan Sivakumar, Simon Gollins, Nhan L Tran, Harshil D Dhruv, Michael E Berens, Alan Bryce, Aleksandar Sekulic, Antoni Ribas, Jeffrey M Trent, Patricia M LoRusso, Muhammed Murtaza
bioRxiv 126839; doi: https://doi.org/10.1101/126839
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Evaluation of pre-analytical factors affecting plasma DNA analysis
Havell Markus, Tania Contente-Cuomo, Winnie S Liang, Mitesh J Borad, Shivan Sivakumar, Simon Gollins, Nhan L Tran, Harshil D Dhruv, Michael E Berens, Alan Bryce, Aleksandar Sekulic, Antoni Ribas, Jeffrey M Trent, Patricia M LoRusso, Muhammed Murtaza
bioRxiv 126839; doi: https://doi.org/10.1101/126839

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