TY - JOUR
T1 - Use of FFPE-derived DNA in next generation sequencing
T2 - DNA extraction methods
AU - Mcdonough, Samantha J.
AU - Bhagwate, Aditya
AU - Sun, Zhifu
AU - Wang, Chen
AU - Zschunke, Michael
AU - Gorman, Joshua A.
AU - Kopp, Karla J.
AU - Cunninghamid, Julie M.
N1 - Publisher Copyright:
© 2019 McDonough et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/4
Y1 - 2019/4
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85064431694&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85064431694&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0211400
DO - 10.1371/journal.pone.0211400
M3 - Article
C2 - 30973937
AN - SCOPUS:85064431694
SN - 1932-6203
VL - 14
JO - PloS one
JF - PloS one
IS - 4
M1 - e0211400
ER -