Project description:Interventions: Group 1: Blood and sputum samples as well as paraffin embedded tumour tissue from patients with microsatellite stable colorectal cancer shall be analysed before therapy and over time to establish and validate hotspot mutation and somatic copy number variant (SCNAs) analysis. We therefore need the following samples:
- Two Cell-Free DNA BCT CE Streck tubes with 8 ml blood per sampling for preparation of plasma-DNA.
- One sputum tube (only at study inclusion).
- FFPE tissue samples from the primary tumor (from initial surgery)
Group 2: Blood samples of tumor-free control persons shall be tested for hotspot mutations and somatic copy number variants (SCNAs) to identify technical artefacts and improve our protocols. We therefore need the following samples:
- Two Cell-Free DNA BCT CE Streck tubes with 8 ml blood per sampling for preparation of plasma-DNA.
Primary outcome(s): Identification of tumorspecific SCNAs in plasma samples of colorectal cancer patients
Study Design: Allocation: ; Masking: ; Control: ; Assignment: ; Study design purpose: other
Project description:In this study, we compared the two long-read sequencing platforms, namely the single-molecule real-time sequencing by Pacific Biosciences and nanopore sequencing by Oxford Nanopore Technologies, for the analysis of cell-free DNA from plasma. Artificial mixtures of sonicated human and mouse DNA at different sizes were sequenced with the two platforms.
Project description:The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of fragmentomics. This study attempted to further characterize the end structure of plasma DNA.
Project description:The structure of broken DNA ends is a critical determinant of the pathway used for DNA double strand break (DSB) repair. Here, we develop an approach, hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1-phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3’ overhangs, many of these DNA ends unexpectedly form long 5’ single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify new features of DNA end processing during DSB repair.
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
