{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T12:00:19Z","timestamp":1789214419161,"version":"build-2803163510"},"reference-count":41,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T00:00:00Z","timestamp":1750809600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000048","name":"American Cancer Society","doi-asserted-by":"publisher","award":["RSG-17-044-01-LIB"],"award-info":[{"award-number":["RSG-17-044-01-LIB"]}],"id":[{"id":"10.13039\/100000048","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/biom15070927","type":"journal-article","created":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T03:53:02Z","timestamp":1750823582000},"page":"927","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Impact of Long-Term Plasma Storage on Cell-Free DNA Epigenetic Biomarker Studies"],"prefix":"10.3390","volume":"15","author":[{"given":"Jianming","family":"Shao","sequence":"first","affiliation":[{"name":"Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thao","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2714-8940","authenticated-orcid":false,"given":"Zejuan","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX 77030, USA"},{"name":"Houston Methodist Research Institute, Houston, TX 77030, USA"},{"name":"Weill Cornell Medical College, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e2325332","DOI":"10.1001\/jamanetworkopen.2023.25332","article-title":"Association of Circulating Tumor DNA Testing Before Tissue Diagnosis With Time to Treatment Among Patients With Suspected Advanced Lung Cancer: The ACCELERATE Nonrandomized Clinical Trial","volume":"6","author":"Law","year":"2023","journal-title":"JAMA Netw. 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