Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

ABSTRACT

The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulphite-based cfDNA methylation profiling. Fifteen tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulphite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

KEYWORDS:

• Liquid biopsy
• cell-free reduced representation bisulfite sequencing (cfRRBS)
• pre-analytical factors
• methylation
• cfDNA

Acknowledgements

We thank all healthy volunteers for participating in this study. A.D.K. and R.V.P. were funded by a predoctoral fellowship from the Research Foundation Flanders (FWO). B.D.W is a senior clinical investigator for the FWO. This project was partially funded by Kom op tegen Kanker, a CRIG (Cancer Research Institute Ghent) Young Investigator Proof-of-Concept grant (to B.D.W.), and by the VIB Grand Challenges Program (Nico Callewaert lab). We thank Peter Stockwell for assistance with mkrrgenome. We thank Charlotte Vandeputte, Eveline Debals, Malaïka Van Der Linden, Thalia Van Laethem, Eveline Vanden Eynde, Kimberly Verniers, Peter Degrave, Dimitri Broucke, Machteld Baetens, Aline Eggermont and Els De Smet for assistance with plasma preparation, cfDNA extraction, library preparation and sequencing. We thank the members of the extracellular RNA quality control consortium (exRNAQC) at Ghent University (Eva Hulstaert, Hetty Helsmoortel, Kimberly Verniers, Francisco Avila Cobos, Anneleen Decock, Eveline Vanden Eynde, Céleste Van der Schueren, Jasper Anckaert, Justine Nuytens, Jill Deleu, Nurten Yigit, Celine Everaert, Jilke De Wilde, Kathleen Schoofs, Gary Schroth, Scott Kuersten, Nele Nijs, Carolina Fierro, Olivier De Wever, An Hendrix, Bert Dhondt, Annouck Philippron) for assistance with experimental setup, data-analysis and interpretation. The computational resources (Stevin Supercomputer Infrastructure) and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by Ghent University, FWO and the Flemish Government – department EWI.

Data availability

Scripts, supporting files and full data analysis is available at https://github.com/rmvpaeme/cfRRBS_tube_study.

Raw data is available at EGA identifier EGAD00001006007. Processed data can be found at ArrayExpress identifier E-MTAB-8858.

The cf-RRBS protocol is available at https://www.protocols.io/private/4098389D37C151C92AA19EC574AD9201.

Disclosure statement

A.D.K is listed as co-inventor in patent application PCT/EP2017/056850 related to the cf-RRBS method. Roche provided the cell-free DNA collection tubes free of charge. Roche was not involved in study design or writing of this manuscript.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Fonds Wetenschappelijk Onderzoek; Fonds Wetenschappelijk Onderzoek; Fonds Wetenschappelijk Onderzoek; Cancer Research Institute Gent; VIB Grand Challenges Program; Kom op tegen Kanker (BE).