Evaluation of pre-analytical factors affecting plasma DNA analysis

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 col...

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Autores principales:	Havell Markus, Tania Contente-Cuomo, Maria Farooq, 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
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/754bebe0d60d4db2a753e90d4a1c1a88
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spelling	oai:doaj.org-article:754bebe0d60d4db2a753e90d4a1c1a882021-12-02T11:41:02ZEvaluation of pre-analytical factors affecting plasma DNA analysis10.1038/s41598-018-25810-02045-2322https://doaj.org/article/754bebe0d60d4db2a753e90d4a1c1a882018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25810-0https://doaj.org/toc/2045-2322Abstract 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.Havell MarkusTania Contente-CuomoMaria FarooqWinnie S. LiangMitesh J. BoradShivan SivakumarSimon GollinsNhan L. TranHarshil D. DhruvMichael E. BerensAlan BryceAleksandar SekulicAntoni RibasJeffrey M. TrentPatricia M. LoRussoMuhammed MurtazaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Havell Markus Tania Contente-Cuomo Maria Farooq 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 Evaluation of pre-analytical factors affecting plasma DNA analysis
description	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.
format	article
author	Havell Markus Tania Contente-Cuomo Maria Farooq 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
author_facet	Havell Markus Tania Contente-Cuomo Maria Farooq 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
author_sort	Havell Markus
title	Evaluation of pre-analytical factors affecting plasma DNA analysis
title_short	Evaluation of pre-analytical factors affecting plasma DNA analysis
title_full	Evaluation of pre-analytical factors affecting plasma DNA analysis
title_fullStr	Evaluation of pre-analytical factors affecting plasma DNA analysis
title_full_unstemmed	Evaluation of pre-analytical factors affecting plasma DNA analysis
title_sort	evaluation of pre-analytical factors affecting plasma dna analysis
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/754bebe0d60d4db2a753e90d4a1c1a88
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Evaluation of pre-analytical factors affecting plasma DNA analysis

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