# Impact of Long-Term Plasma Storage on Cell-Free DNA Epigenetic Biomarker Studies

## TL;DR

Long-term storage of plasma samples can reduce the quality of cell-free DNA and epigenetic biomarker analysis due to increased DNA contamination.

## Contribution

This study reveals that prolonged plasma storage increases genomic DNA contamination, affecting cfDNA and 5hmC sequencing outcomes.

## Key findings

- Extended plasma storage significantly reduces uniquely mapped reads in 5hmC sequencing.
- Genomic DNA contamination compromises cfDNA fragment integrity and sequencing success.
- gDNA contamination is independently linked to decreased 5hmC sequencing quality in all sample groups.

## Abstract

Impact of long-term plasma storage on biomarker analysis is critical for ensuring data reliability. Cell-free DNA (cfDNA) epigenetic markers, including 5-hydroxymethylcytosine (5hmC), have emerged for disease detection, prognosis, and treatment response. However, the effects of prolonged storage on 5hmC analysis remain unclear. We evaluated the quantity and quality of cfDNA and 5hmC sequencing analyses in 1070 plasma samples stored for up to 14 years from patients with solid tumors and acute myeloid leukemia (AML) and non-cancer individuals. In long-term stored plasma samples, cfDNA yield remained largely stable; however, uniquely mapped reads (UMRs) from 5hmC sequencing were significantly reduced in solid tumor and control samples. Notably, prolonged plasma storage independently contributed to increased genomic DNA (gDNA) contamination in solid tumor and AML samples and significantly correlated with decreased UMRs in control samples. Across all groups, samples with gDNA contamination exhibited significantly reduced UMRs. Furthermore, gDNA contamination independently compromised cfDNA fragment integrity, decreased sequencing library success in solid tumors, and reduced 5hmC sequencing UMRs across all groups. Therefore, extended plasma storage contributes to increased gDNA contamination, compromising cfDNA and 5hmC sequencing quality. Implementing measures to minimize gDNA contamination in long-term plasma storage is crucial for improving downstream cfDNA analysis reliability.