Poster Presentation Australian Epigenetics Alliance Conference 2022

Evaluating DNA recovery efficiency following bisulfite modification from plasma samples submitted for cell free DNA methylation analysis (#123)

Daniel RA Cox 1 2 3 , Boris KL Wong 2 , Eunice Lee 1 3 , Adam Testro 4 5 , Vijayaragaran Muralidharan 1 3 , Alexander Dobrovic 1 2 , Su Kah Goh 1 2
  1. Surgery, University of Melbourne, Melbourne, VIC, Australia
  2. Translational Genomics and Epigenomics Laboratory, University of Melbourne, Melbourne, VIC, Australia
  3. HPB & Liver Transplant Surgery Unit, Austin Health, Melbourne, VIC, Australia
  4. Gastroenterology and Hepatology, Austin Health, Melbourne, VIC, Australia
  5. Medicine, University of Melbourne, Melbourne, VIC, Australia

Background: The detection of methylated templates in cell-free DNA (cfDNA) is increasingly recognised as a valuable, non-invasive tool for diagnosis and prognostication in a range of medical contexts. Significant variations in the recovery of DNA following pre-analytical processes such as cfDNA extraction and sodium bisulfite modification may confound downstream analysis, particularly when accurate quantification of templates is required. Given the wealth of applications for this emerging technology, attention has turned to minimising pre-analytical variables prior to cfDNA methylation analysis.

Methods: We recently described the development of an approach using an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA following the extraction and BISulfite modification steps (CEREBIS). (1)  The strategic placement of cytosine bases in the 180 bp CEREBIS spike-in control enables PCR amplification of the construct by a single primer set both after plasma DNA extraction and following bisulfite modification. Here we report the application of this technique in 107 consecutive patient plasma samples submitted for quantitative cfDNA methylation analysis.

Results: The mean recovery of cfDNA (estimated using CEREBIS), following extraction and bisulfite modification, was 37% +/- 7%. 9/107 samples (8.4%) were found to be outside of control limits, where the recovery of CEREBIS indicated significant differences in the efficiency of sample pre-analytical processing. Recognition of these out-of-control samples precluded subsequent molecular analysis.

Discussion: Monitoring of CEREBIS recovery has two key advantages. Firstly, it can be used to identify samples breaching control limits. Secondly, CEREBIS can assist in further assuring that any observed biological effect in serial samples analysed from a single patient is likely true and unlikely to be related to the pre-analytical technical factors in cfDNA extraction and bisulfite modification. Implementation of data-driven quality control measures, such as the one described, has the potential to improve the quality of liquid biopsy methylation analysis, interpretation and reporting.

  1. (1) Goh SK, Cox DRA, Wong BKL, Musafer A, Witkowski T, Do H, Muralidharan V, Dobrovic A. A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification. Clin Chem 2021; 67:1201-9.