Evaluating DNA recovery efficiency following bisulphite modification from plasma samples submitted for cell-free DNA methylation analysis

ABSTRACT

The detection of methylated templates in cell-free DNA (cfDNA) is increasingly recognized as a valuable, non-invasive tool for diagnosis, monitoring and prognostication in a range of medical contexts. The importance of controlling pre-analytical conditions in laboratory workflows prior to cfDNA quantification is well-established. Significant variations in the recovery of DNA following processes such as cfDNA extraction and sodium bisulphite modification may confound downstream analysis, particularly when accurate quantification of templates is required. Given the wealth of potential applications for this emerging molecular technology, attention has turned to the requirement to recognize and minimize pre-analytical variables prior to cfDNA methylation analysis. We recently described the development of an approach using an exogenous DNA construct to evaluate the recovery efficiency of cfDNA following the extraction and bisulphite modification steps (CEREBIS). Here, we report our experience in the practical application of this technique in 107 consecutive patient plasma samples submitted for quantitative cfDNA methylation analysis. The mean recovery of cfDNA (as estimated using cerebis), following extraction and bisulphite modification, was 37% ± 7%. Nine (8.4%) of the 107 samples were found to be outside of control limits, where the recovery of cerebis indicated significant differences in the efficiency of the pre-analytical processing of these samples. Recognition of these out-of-control samples precluded subsequent molecular analysis. 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.

KEYWORDS:

• DNA methylation
• quality control
• pre-analytical
• DNA controls
• digital PCR
• cell-free DNA

Abbreviations

AT, ambient temperature; CEREBIS, Construct to Evaluate Recovery Efficiency after cell-free DNA extraction and Bisulfite modification; cfDNA, cell-free DNA; ddPCR, droplet digital PCR; DNA, deoxyribonucleic acid.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author on application upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2022.2091821

Additional information

Funding

DC: Ashley Johnson Scholarship (Victorian Liver Transplant Unit) and the Research Training Program Scholarship (University of Melbourne). AD: National Breast Cancer Foundation of Australia; National Health and Medical Research Council, Australia; University of Melbourne Department of Surgery; The University of Melbourne.