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.
Project description:Comparitive proteomics analyses of different cell types of blood i.e. platelets, neutrophils, monocytes and CD4 derived from the Gray platelet syndrome patients and their respective controls using TMT10plex reagents.
Project description:Here we used Illumina NGS for high-throughput profiling of the DNA methylome(ERRBS) and hydroxymethylome(hMe-Seal) of primary tumor samples with Acute Myeloid Leukemia(AML). The data can be used to compare hydroxymethylation and methylation patterns from different AML subtypes and normal bone marrow samples. We have sequenced 4 subtypes of AML with hydroxymethylation decrease and 1 subtype with no decrease. We have sequenced 2-5 primary tumor samples for each subtype, and comprated the epigenomic profiles ( ERRBS and hMe-Seal ) of hydroxymethylation deficient subtypes to the control subtype and normal bone marrow samples.
Project description:We sequenced total RNA from whole blood samples of 27 wild gray wolves from Yellowstone National Park. Gene expression level analysis of both male and female wolves, ranging from ages 0.8-8.8 years.
Project description:In the present study, we applied two whole-genome sequencing techniques (WGBS/oxBS and hMe-Seal) to detect 5hmC (and 5mC) changes during the differentiation of the human SGBS preadipocyte cell line to mature adipocytes. As technical and biological validation we performed BS and oxBS followed by 450k array analysis. RNA-seq data was performed in parallel to study transcriptional changes associated with differential hydroxymethylation. In human white adipose tissue (WAT) hydroxymethylation (hME-Seal) was characterized in comparison with histone modifications and acNEIL1 binding (ACT-seq).
Project description:Investigations of 5-hydroxymethylcytosine (5hmC) in biologically and clinically samples and models with low cell numbers have been hampered by the low sensitivity and reproducibility using current 5hmC mapping approaches. Here, we develop a selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we profiled the dynamics of 5hmC across different stages of mouse hematopoietic differentiation. Additionally, applying nano-hmC-Seal to the hematopoietic multipotent progenitor cells in an acute myeloid leukemia (AML) mouse model, we identified leukemia-specific, differentially hydroxymethylated regions that harbor previously reported and as-yet-unidentified functionally relevant factors. The change of 5hmC patterns in AML strongly correlates with the altered gene expression on a global scale. Together, our new approach offers a highly sensitive and robust method to study and detect DNA methylation dynamics from in vivo model and clinical samples. Selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells