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Licensed Unlicensed Requires Authentication Published by De Gruyter July 16, 2020

A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers

  • Shengnan Jin , Qian Ye , Yanping Hong , Wenqing Dai , Chengliang Zhang , Weihao Liu , Ying Guo , Dewen Zhu , Zhengzheng Zhang , Shiliang Chen , Yourong Wang , Dandan Li , Wen Ma , Zhengquan Yang , Jinlei Li , Zhihai Zheng , Ju Luan , Xiaoli Wu , Feizhao Jiang , Chang Xu EMAIL logo and Chunming Ding EMAIL logo



Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.


We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.


The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.


Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

Corresponding authors: Chunming Ding, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, Wenzhou, P.R. China; Key Laboratory of Laboratory Medicine, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, PR China, Phone: +86 577 86690891, Fax: +86 577 86699779, E-mail: ; and Chang Xu, Department of Colorectal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Zhejiang, PR China, E-mail:
Shengnan Jin and Qian Ye are joint first authors.

Award Identifier / Grant number: 81672922

Funding source: Medical Health Science and Technology Project of Zhejiang Provincial Health Commission

Award Identifier / Grant number: 2017174160

Funding source: Wenzhou Science and Technology Bureau

Award Identifier / Grant number: Y20160044

Award Identifier / Grant number: 2018ZY005

Funding source: High-Level Innovation Team of Universities in Zhejiang Province

Award Identifier / Grant number: 604090352/610

Funding source: Innovation Discipline of Zhejiang Province in Nucleic Acid Molecular Diagnostics

Award Identifier / Grant number: 437201702G

Funding source: Key Discipline of Zhejiang Province in Medical Technology

Award Identifier / Grant number: 437601607


We would like to thank all the patients who participated in the studay and all the support from the doctors and nurses for the collection of samples.

  1. Research funding: This work was supported by the National Natural Sciences Foundation of China (CD: No. 81672922), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (CX: No. 2017174160), Wenzhou Science and Technology Bureau (CD: No. 2018ZY005; CX: No. Y20160044), High-Level Innovation Team of Universities in Zhejiang Province (CD, No. 604090352/610), Innovation Discipline of Zhejiang Province in Nucleic Acid Molecular Diagnostics (CD: No. 437201702G) and Key Discipline of Zhejiang Province in Medical Technology (Fist Class, Category A) (CD: No. 437601607).

  2. Author contributions: Shengnan Jin, Chang Xu and Chunming Ding: design of the project, data interpretation, writing of the manuscript; Qian Ye: performance of lab work and interpretation of data, writing of the manuscript; Zhengzheng Zhang, Shiliang Chen, Yourong Wang, Dandan Li, Wen Ma, Yanping Hong, Dewen Zhu, Wenqing Dai, Chengliang Zhang, Weihao Liu and Ying Guo: sample collection and clinical database curation; Zhengquan Yang, Jinlei Li, Ju Luan, Xiaoli Wu, Feizhao Jiang and Zhihai Zheng: patient enrollment. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The study was approved by the Clinical Research Ethics of the First Affiliated Hospital of Wenzhou Medical University.


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Supplementary material

The online version of this article offers supplementary material (

Received: 2020-03-11
Accepted: 2020-06-22
Published Online: 2020-07-16
Published in Print: 2021-01-26

© 2020 Walter de Gruyter GmbH, Berlin/Boston

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