The University of Toledo Translation Journal of Medical Sciences 
Pulmonology Abstract, Department of Medicine Research Symposium UTJMS 2023 May 5; 11(1):e1-e1 

Use of Internal Standards to Develop 
Improved Methylation Detection in cfDNA 
Erin L. Crawford1*, Daniel J. Craig1, James G. Herman1, James C. Willey1

1Division of Pulmonology and Critical Care Medicine, Department of Medicine, The 
University of Toledo, Toledo, OH 43614 

*Corresponding author: Erin.crawford@rockets.utoledo.edu

Published: 05 May 2023 

Detection of aberrant levels and patterns of methylation in circulating free genomic DNA (cfDNA) may 
be useful in predicting the presence of malignancy in a particular tissue or in monitoring known 
malignancies for recurrence or treatment resistance. The goal of this study is to develop methods to 
improve accurate and sensitive detection of methylation in cfDNA utilizing synthetic spike-in internal 
standards (IS) that enable target- and assay-specific limits of detection and control for technical error. 
Initial testing of an off-the-shelf IS yielded promising results but, technical error increased as 
methylation increased, likely due to suboptimal IS design and experimental conditions.  Next, a mixture 
of optimized IS for five targets known to be altered in cancer, SOX2, CDO1, TAC1, SOX17, and 
HOXA7, was created. Each IS was designed to be spiked into DNA, bisulfite-treated and measured in 
either hybrid capture or amplicon-based next generation sequencing libraries.  Sequence changes (either 
A>T or T>A to avoid changes caused by bisulfite treatment) were introduced approximately every 50 bp 
to allow the IS to be distinguished from endogenous DNA.  IS were cloned into plasmids, linearized, 
quantified and combined to create the IS mix.  This IS mixture will be combined with a set of reference 
human genomic DNA samples obtained from the National Institute of Standards and Technology known 
to have differing levels of methylation such that the ratio of IS mixture:genomic DNA varies. These will 
be bisulfite-treated, used to prepare amplicon libraries and sequenced. Methylation, technical error and 
limit of detection will be assessed.

https://dx.doi.org/10.46570/utjms.vol11-2023-776 

https://dx.doi.org/10.46570/utjms.vol11-2023-776
mailto:Erin.crawford@rockets.utoledo.edu