Project description:Cell-free DNA (cfDNA) fragmentation patterns encode biologically and clinically relevant information beyond fragment length, reflecting nuclease activity, chromatin organization, and tissue of origin. Fragmentomics has therefore emerged as a promising strategy to enhance circulating tumor DNA (ctDNA) detection, particularly in cancers with low tumor fractions. However, most existing approaches are optimized for short-read sequencing, limiting their applicability to third-generation platforms. Here, we present FLARE (Fragmentation and Long-read Analysis of Regulatory Epigenetics), an integrated and scalable fragmentomics pipeline specifically optimized for Oxford Nanopore long-read sequencing. FLARE preserves native cfDNA fragment ends and enables the simultaneous analysis of copy number alterations, tumor fraction estimation, methylation-derived signals, fragment length distributions, and 5′ end-motif profiles.
Project description:Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ), schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ, e.g., PRODH and DGCR8. However, the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development, we carried out RNA-seq analysis to investigate gene expression in differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subject to RNA sequencing. Using a systems level analysis, differentially expressed genes/gene-modules and pathway of interests were identified. We observed ~2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value < 0.05, 36 of which reached a false discovery rate < 0.05). Outside of the deleted region, 745 genes showed significant differences in expression between SZ and control neurons (p<0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways, such as apoptosis, cell cycle and survival, and MAPK signaling in the SZ and SAD samples.
Project description:Background: To better understand the role DNA methylation plays in regulating gene expression in the developng heart and furthermore the role it plays in heart development we performed genome wide DNA methylation profiling of embryonic hearts at embryonic day (E)11.5 and E14.5 using methyl sensitive tiny fragment enrichment coupled with massive parallel sequencing by using the methyl-sensitive restriction enzyme HpyCH4IV, recognition site 'ACGT'. Results: We found that global methylation remains stable at analyzed 'ACGT' sites (1.64 million site) in developing hearts between E11.5 and E14.5. However, differential methylation was identified at individual loci enriched at genes involved in heart development suggesting a role for DNA methylation in the developing heart. Used Methyl Sensitive Tiny Fragment Enrichment/Massive Parallel Sequencing (MSFE/MPS) to assay methylation at 'ACGT' sites throughout the genome and generate a developmental profile of DNA methylation in the embryonic heart and to identify differences between developing mouse hearts at E11.5 and E14.5.
Project description:Genomic DNA from 39 recq4 Col x Ler F2 individuals was extracted using the CTAB method. Equal amounts of DNA from these 39 plants were pooled and nine micrograms of gDNA from each pool was used to generate Nanopore sequencing libraries with the Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced independently using PromethION (BGI, Hong Kong).
Project description:As an essential regulator of higher-order chromatin structures, CTCF is a highly conserved DNA binding protein with a central DNA-binding domain of 11 tandem zinc fingers, which are flanked by N- and C-terminal domains of intrinsically disordered regions. The N-terminal domain interacts with cohesin complex and the central ZF domains recognize a large range of diverse genomic sites. However, the function of C-terminal unstructured domain remains less understood. Here, we found that deletion of C-terminal fragment of 116 amino acid increases CTCF-DNA binding. By genetic dissection of this fragment, we uncovered a negatively-charged region responsible for repressing CTCF binding to DNA. Thus, the unstructured C-terminal domain play an important role in maintaining proper CTCF-DNA interactions.
Project description:As an essential regulator of higher-order chromatin structures, CTCF is a highly conserved DNA binding protein with a central DNA-binding domain of 11 tandem zinc fingers, which are flanked by N- and C-terminal domains of intrinsically disordered regions. The N-terminal domain interacts with cohesin complex and the central ZF domains recognize a large range of diverse genomic sites. However, the function of C-terminal unstructured domain remains less understood. Here, we found that deletion of C-terminal fragment of 116 amino acid increases CTCF-DNA binding. By genetic dissection of this fragment, we uncovered a negatively-charged region responsible for repressing CTCF binding to DNA. Thus, the unstructured C-terminal domain play an important role in maintaining proper CTCF-DNA interactions.
Project description:This dataset contains Xdrop followed by oxford nanopore long read sequencing performed in target tRNA gene deletion (t8) and intergenic region deletion (i50) clones in HepG2 . By applying de novo assembly based approach to Xdrop-LRS data, we identified Cas9-induced on-target genomic alteration.
Project description:Genomic DNA from 55 wild type Col x Ler F2 individuals was extracted using the CTAB method. Equal amounts of DNA from these 55 plants were pooled into two groups (pool 1 = 4 plants; pool 2 = 51 plants), and nine micrograms of gDNA from each pool was used to generate Nanopore sequencing libraries with the Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced independently using PromethION (BGI, Hong Kong).