Project description:We evaluated whether targeted next-generation sequencing (NGS) using the Ion Torrent Personal Genome Sequencer of cfDNA could identify prognostic or predictive factors for overall survival (OS) or progression free survival (PFS) within a large cohort of patients with advanced lung adenocarcinoma enrolled in the GALAXY-1 trial.
Project description:We performed all stimulatory experiments using THP1 cell line as a representative of primary human monocytes to show fundamental role of the cfDNA in healthy organisms. The experiments were conducted in duplicates using plasma containing cfDNA (NP) and the reference one with cfDNA removed by DNase (TP) to recognize unequivocally the effect of plasma cfDNA on transcriptome and proteome of monocytes. We used native human plasma samples obtained from healthy volunteers with no animal serum addition to cultivation medium in order to avoid the presence of uncharacterized animal cfDNA in the experiments.
Project description:The unique chromatin structure at telomeres protects the ends of our chromosomes from being recognized as DNA damage and preserves genome integrity by preventing recombination. Here, we demonstrate that circulating cell-free DNA (cfDNA) from plasma can be used to map chromatin structure at telomeres. Using an alignment-free approach to overcome challenges in characterizing repetitive regions of DNA using sequencing, we find that the telomeric 6-mer repeats (TTAGGG/CCCTAA) are the most abundant circulating 6-mers in cfDNA. Telomeric sequences in cfDNA contain subnucleosomal footprints distinct from the rest of the genome, arising from specific cleavages in the C-rich strand. To map nucleosomal footprints from telomeric sequences in cfDNA, we performed long-read sequencing. We identified a nucleosome repeat length of 145 bp for telomeric sequences, which is significantly shorter than the repeat length of ~170 bp for the rest of the genome. The abundance of cfDNA telomeric footprints decreases with age, and this decline is exacerbated by Dyskeratosis Congenita (DC), a telomere biology disorder. DC also alters subtelomeric chromatin accessibility, as determined from cfDNA, allowing us to capture genome-wide effects of shortened telomeres non-invasively. Promoter subnucleosome enrichment (PSE) from cfDNA identifies DC-specific gene signatures that reflect disease states observed in transcriptomic studies. Notably, DC-specific gene signatures correlate with decreased cfDNA telomere footprint abundance and show enrichment of genes towards the ends of chromosomes. In summary, cfDNA provides an unexpected means to map telomere chromatin state and the impact of telomere shortening on the rest of the genome, offering a non-invasive way to track gene expression changes in DC.
Project description:Heterogeneity in DNA methylation status may exist between primary tumors and cfDNA. We compared the genome-wide DNA methylation status between FNA sample of pancreatic tumor and cfDNA samples from the same patients. MBD-sequencing (seq) was performed in paired samples of FNA and cfDNA from two patients. The majority of DNA methylation peaks overlapped in FNA and cfDNA samples (82.6% and 82.9% in patients #1 and #73, respectively). DNA methylation levels of each gene in FNA and cfDNA samples were highly correlated with each other (r=0.98 and 0.97 in samples 1 and 73, respectively)
Project description:We aimed to clarify the cell-free DNA (cfDNA) physiological role. We exposed THP1 cells to plasma from healthy individuals with and without cfDNA and compared their transcriptomes and proteomes.
Project description:Liquid biopsies hold significant potential for the non-invasive diagnostics of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial pre-analytical variations. In this work, we are taking a step towards standardizing the pre-analytical procedures of blood collection for subsequent co-isolation of plasma cfDNA and EVs from a single blood collection tube. We compare effects of blood preservation tubes and storage to enable proteomic profiling of resulting EVs in addition to cfDNA extraction and sequencing. Following a stringent method of large EV (lEV) and small EV (sEV) isolation, consisting of differential ultracentrifugation and size exclusion chromatography, we evaluate protein concentration, particle number, quality and integrity of the isolated EVs. Subsequent proteomic analyses of EV isolates unravel the complexity of the respective tube proteomes allowing the interpretation of EV origins as well as contamination sources. While ACD-A and Citrate tubes demonstrate satisfying results in preservation of EV proteomes, only Streck RNA®, Norgen® and PAX® tubes can preserve high cfDNA purity for up to 7 days. When aiming for multi-omics analyses, Streck RNA® tubes show the most stable performance across tested parameters for both bioanalytes. Furthermore, we find more variability in protein composition in sEVs than in lEVs after 7 days of storage; thus, sEVs might be more susceptible to storage effects. Our clinically applicable workflow provides the basis for informed choice of liquid biopsy tubes along with a ready-to-use protocol to retrieve both genomic and EV proteomic biomarker information for multi-omics biomarker-based liquid biopsy studies.
Project description:The genome-wide analysis of cfDNA fragmentation patterns in DENQCMs and maternal plasma was performed by deep sequencing using Illumina Novaseq to confirm their biological characteristics. We first performed deep whole genome sequencing of the cfDNA extracted from DENQCMs and maternal plasma cfDNA. Then we analyzed the sequencing data and compared various characteristics of cfDNA fragmentation patterns, such as dinucleotide composition, nucleosome protection length, and nucleosome occupancy based on a windowed protection score (WPS), to the submitter-provided processed data from a healthy individual IH01 (GEO accession GSM1833276).
Project description:We provide the first in vivo evidence of global and local chromatin changes in human aging by analyzing cfDNA from the blood of individuals of different age groups. Our results show that nucleosome signals inferred from cfDNA are consistent with the redistribution of heterochromatin observed in cellular senescence and aging in other model systems. It also revealed age and deteriorating health status correlate with a low-level enrichment of signals from cells in the thyroid gland. In addition, we detected an overall nucleosome loss at several genomic locations, such as transcription start and termination sites, 5’UTR of L1HS retrotransposons and dimeric AluY elements with age.