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Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE...

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Autores principales: McDonough, Samantha J., Bhagwate, Aditya, Sun, Zhifu, Wang, Chen, Zschunke, Michael, Gorman, Joshua A., Kopp, Karla J., Cunningham, Julie M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459541/
https://www.ncbi.nlm.nih.gov/pubmed/30973937
http://dx.doi.org/10.1371/journal.pone.0211400
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author McDonough, Samantha J.
Bhagwate, Aditya
Sun, Zhifu
Wang, Chen
Zschunke, Michael
Gorman, Joshua A.
Kopp, Karla J.
Cunningham, Julie M.
author_facet McDonough, Samantha J.
Bhagwate, Aditya
Sun, Zhifu
Wang, Chen
Zschunke, Michael
Gorman, Joshua A.
Kopp, Karla J.
Cunningham, Julie M.
author_sort McDonough, Samantha J.
collection PubMed
description Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS.
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spelling pubmed-64595412019-05-03 Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods McDonough, Samantha J. Bhagwate, Aditya Sun, Zhifu Wang, Chen Zschunke, Michael Gorman, Joshua A. Kopp, Karla J. Cunningham, Julie M. PLoS One Research Article Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS. Public Library of Science 2019-04-11 /pmc/articles/PMC6459541/ /pubmed/30973937 http://dx.doi.org/10.1371/journal.pone.0211400 Text en © 2019 McDonough et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
McDonough, Samantha J.
Bhagwate, Aditya
Sun, Zhifu
Wang, Chen
Zschunke, Michael
Gorman, Joshua A.
Kopp, Karla J.
Cunningham, Julie M.
Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title_full Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title_fullStr Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title_full_unstemmed Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title_short Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods
title_sort use of ffpe-derived dna in next generation sequencing: dna extraction methods
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459541/
https://www.ncbi.nlm.nih.gov/pubmed/30973937
http://dx.doi.org/10.1371/journal.pone.0211400
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