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2af0d6d0-7214-440c-a3ff-7189eefec97a - samples

ABSTRACT: Plasma from lung cancer patients from EDTA tubes was fractionated using size exclusion chromatography. Fractions 1-5, 7-11, 12-15, 16-20 were pooled, cfDNA was extracted from the fractions and paired unfractionated samples and PE150bp sequencing was performed on an Illumina Novaseq S4 flowcell. Samples are provided as raw reads without any prior processing.

PROVIDER: EGAD00001009780 | EGA |

REPOSITORIES: EGA

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

Project description:Background 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 bisulfite-based cfDNA methylation profiling. Methods 15 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 bisulfite 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. Results 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. Conclusion 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.

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Project description:Background: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin. Methods: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who later were diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features. Results: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay. Conclusions: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analyzable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.

2023-11-14 | GSE230027 | GEO

Circulating-cell free DNA pooled samples for epigenome-wide discovery of methylation biomarkers for colorectal cancer screening

Project description:We explored the differential methylation patterns found in cfDNA between healthy controls (individuals with no colorectal findings, NCF) and patients with advanced neoplasia (advanced adenomas and colorectal cancer, AA andCRC) using pooled samples, to determine if pooled serum cfDNA samples can be used to search for non-invasive methylation biomarkers, as an affordable and efficient alternative to tissue biopsy. cfDNA was extracted from serum samples and methylation measurements were assessed with the Infinium MethylationEPIC BeadChip. Data was mainly preprocessed and analyzed with R/Bioconductor packages.

2018-04-17 | GSE110185 | GEO

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