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Deep oncopanel sequencing reveals fixation time- and within block position-dependent quality degradation in FFPE processed samples

SEQC2 Oncopanel Sequencing Working Group, View ORCID ProfileYifan Zhang, Thomas M. Blomquist, Rebecca Kusko, Daniel Stetson, Zhihong Zhang, Lihui Yin, Robert Sebra, Binsheng Gong, Jennifer S. LoCoco, Vinay K. Mittal, Natalia Novoradovskaya, Ji-Youn Yeo, Nicole Dominiak, Jennifer Hipp, Amelia Raymond, Fujun Qiu, Hanane Arib, Melissa L. Smith, Jay E. Brock, Daniel H. Farkas, Daniel J. Craig, Erin L. Crawford, Dan Li, Tom Morrison, Nikola Tom, Wenzhong Xiao, Mary Yang, View ORCID ProfileChristopher E. Mason, Todd A. Richmond, Wendell Jones, Donald J. Johann Jr, Leming Shi, Weida Tong, James C. Willey, Joshua Xu
doi: https://doi.org/10.1101/2021.04.06.438687
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
Yifan Zhang
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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  • ORCID record for Yifan Zhang
Thomas M. Blomquist
2(formerly) Department of Pathology, College of Medicine and Life Sciences, The University of Toledo, Toledo, OH 43614, USA
3Lucas County Coroner’s Office, 2595 Arlington Ave., Toledo, OH 43614, USA
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Rebecca Kusko
4Immuneering Corporation, One Broadway, 14th Floor, Cambridge, MA 02142, USA
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Daniel Stetson
5Astrazeneca Pharmaceuticals, 35 Gatehouse Dr, Waltham, MA 02451, USA
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Zhihong Zhang
6Research and Development, Burning Rock Biotech, Shanghai 201114, China
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Lihui Yin
7(formerly) Pathology and Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
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Robert Sebra
8Icahn Institute and Dept. of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai, 1425 Madison Ave., New York, NY 10029, USA
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Binsheng Gong
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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Jennifer S. LoCoco
9Illumina Inc., 5200 Illumina Way, San Diego, CA 92122, USA
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Vinay K. Mittal
10Thermo Fisher Scientific, 110 Miller Ave., Ann Arbor, MI 48104, USA
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Natalia Novoradovskaya
11Agilent Technologies, 11011 N Torrey Pines Rd., La Jolla, CA 92037, USA
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Ji-Youn Yeo
12Department of Pathology, University of Toledo, 3000 Arlington Ave., Toledo, OH 43614, USA
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Nicole Dominiak
12Department of Pathology, University of Toledo, 3000 Arlington Ave., Toledo, OH 43614, USA
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Jennifer Hipp
13Department of Pathology, Strata Oncology, Inc., Ann Arbor, MI 48103, USA
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Amelia Raymond
5Astrazeneca Pharmaceuticals, 35 Gatehouse Dr, Waltham, MA 02451, USA
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Fujun Qiu
6Research and Development, Burning Rock Biotech, Shanghai 201114, China
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Hanane Arib
8Icahn Institute and Dept. of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai, 1425 Madison Ave., New York, NY 10029, USA
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Melissa L. Smith
8Icahn Institute and Dept. of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai, 1425 Madison Ave., New York, NY 10029, USA
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Jay E. Brock
14Pathology and Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
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Daniel H. Farkas
14Pathology and Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
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Daniel J. Craig
15Department of Medicine, College of Medicine and Life Sciences, The University of Toledo, Toledo, OH 43614, USA
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Erin L. Crawford
15Department of Medicine, College of Medicine and Life Sciences, The University of Toledo, Toledo, OH 43614, USA
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Dan Li
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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Tom Morrison
16Accugenomics, Inc., 1410 Commonwealth Drive, Suite 105, Wilmington, NC 20403, USA
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Nikola Tom
17Center of Molecular Medicine, Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
18EATRIS ERIC- European Infrastructure for Translational Medicine, De Boelelaan 1118, 1081 HZ Amsterdam, The Netherlands
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Wenzhong Xiao
19Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
20Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA
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Mary Yang
21Department of Information Science, University of Arkansas at Little Rock, 2801 S. Univ. Ave., Little Rock, AR 72204, USA
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Christopher E. Mason
22Department of Physiology and Biophysics, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA
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  • ORCID record for Christopher E. Mason
Todd A. Richmond
23Market & Application Development Bioinformatics, Roche Sequencing Solutions Inc., 4300 Hacienda Dr., Pleasanton, CA 94588, USA
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Wendell Jones
24Q2 Solutions - EA Genomics, 5927 S Miami Blvd., Morrisville, NC 27560, USA
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Donald J. Johann Jr
25Winthrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, 4301 W Markham St., Little Rock, AR 72205, USA
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Leming Shi
26State Key Laboratory of Genetic Engineering, School of Life Sciences and Shanghai Cancer Hospital/Cancer Institute, Fudan University, Shanghai 200438, China
27Human Phenome Institute, Fudan University, Shanghai 201203, China
28Fudan-Gospel Joint Research Center for Precision Medicine, Fudan University, Shanghai 200438, China
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Weida Tong
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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James C. Willey
29Departments of Medicine, Pathology, and Cancer Biology, College of Medicine and Life Sciences, University of Toledo Health Sciences Campus, 3000 Arlington Ave., Toledo, OH 43614, USA
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  • For correspondence: james.willey2@utoledo.edu joshua.xu@fda.hhs.gov
Joshua Xu
1Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
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  • For correspondence: james.willey2@utoledo.edu joshua.xu@fda.hhs.gov
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Abstract

Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized normal cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. Tissue sections that failed more frequently showed low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces were more likely to show FFPE-specific errors, akin to “edge effects” seen in histology, and the depth of formalin damage was related to fixation time. To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 07, 2021.
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Deep oncopanel sequencing reveals fixation time- and within block position-dependent quality degradation in FFPE processed samples
SEQC2 Oncopanel Sequencing Working Group, Yifan Zhang, Thomas M. Blomquist, Rebecca Kusko, Daniel Stetson, Zhihong Zhang, Lihui Yin, Robert Sebra, Binsheng Gong, Jennifer S. LoCoco, Vinay K. Mittal, Natalia Novoradovskaya, Ji-Youn Yeo, Nicole Dominiak, Jennifer Hipp, Amelia Raymond, Fujun Qiu, Hanane Arib, Melissa L. Smith, Jay E. Brock, Daniel H. Farkas, Daniel J. Craig, Erin L. Crawford, Dan Li, Tom Morrison, Nikola Tom, Wenzhong Xiao, Mary Yang, Christopher E. Mason, Todd A. Richmond, Wendell Jones, Donald J. Johann Jr, Leming Shi, Weida Tong, James C. Willey, Joshua Xu
bioRxiv 2021.04.06.438687; doi: https://doi.org/10.1101/2021.04.06.438687
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Deep oncopanel sequencing reveals fixation time- and within block position-dependent quality degradation in FFPE processed samples
SEQC2 Oncopanel Sequencing Working Group, Yifan Zhang, Thomas M. Blomquist, Rebecca Kusko, Daniel Stetson, Zhihong Zhang, Lihui Yin, Robert Sebra, Binsheng Gong, Jennifer S. LoCoco, Vinay K. Mittal, Natalia Novoradovskaya, Ji-Youn Yeo, Nicole Dominiak, Jennifer Hipp, Amelia Raymond, Fujun Qiu, Hanane Arib, Melissa L. Smith, Jay E. Brock, Daniel H. Farkas, Daniel J. Craig, Erin L. Crawford, Dan Li, Tom Morrison, Nikola Tom, Wenzhong Xiao, Mary Yang, Christopher E. Mason, Todd A. Richmond, Wendell Jones, Donald J. Johann Jr, Leming Shi, Weida Tong, James C. Willey, Joshua Xu
bioRxiv 2021.04.06.438687; doi: https://doi.org/10.1101/2021.04.06.438687

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