Project description:To unravel the fine architecture of neocentromeres found in three well-differentiated liposarcoma (WDLPS) cell lines as patchworks of multiple short amplified sequences, disclosing a much more higher complexity than previously reported. Next generation sequencing data (WGS, RNA-seq, CENP-A/ChIP-seq) are available at the Sequence Read Archive (BioProject ID: PRJNA378952).
Project description:Agilent whole exome hybridisation capture was performed on genomic DNA derived from Chondrosarcoma cancer and matched normal DNA from the same patients. Next Generation sequencing performed on the resulting exome libraries and mapped to build 37 of the human reference genome to facilitate the identification of novel cancer genes. Now we aim to re find and validate the findings of those exome libraries using bespoke pulldown methods and sequencing the products.
Project description:In order to map levels of genome-wide HP1a occupancy we applied DamID (van Steensel & Henikoff, Nat Biotech, 2000; PMID: 10748524) in combination with next-generation sequencing of methylated GATC fragments. Mapping by next-generation sequencing makes it possible to examine heterochromatic regions that were not covered by earlier datasets which were generated using microarrays.
Project description:We developed Del-Read, an algorithm targeting medium-sized deletions (6-100 BPs) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homology mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-Read, we identified numerous novel germline and somatic MMEJ-dels in Beat AML and TCGA-breast datasets. Validation in 500 healthy individuals confirmed their presence.
Project description:Objectives: To perform long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors. We aim to discover new transcripts and protein isoforms expressed during immune responses to diverse pathogens. Methods: PBMCs were exposed to four microbial stimuli for 24 hours: the TLR4 ligand lipopolysaccharide (LPS), the TLR3 ligand Poly(I:C), heat-inactivated Staphylococcus aureus, Candida albicans, and RPMI medium as negative controls. Long-read sequencing (PacBio) of one donor and secretome proteomics and short-read sequencing of five donors were performed. IsoQuant was used for transcriptome construction, Metamorpheus/FlashLFQ for proteome analysis, and Illumina short-read 3’-end mRNA sequencing for transcript quantification. Results: Long-read transcriptome profiling reveals the expression of novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. We observe widespread loss of intron retention as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. In general, RNA expression differences did not result in differences in the amounts of secreted proteins. Interindividual differences in the proteome were larger than the differences between stimulated and unstimulated PBMCs. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Conclusion: Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.
Project description:Transcribed regions in adult temporal lobe, hippocampus and frontal lobe were assesed by strand specific next generation sequencing of polyA RNA. Strand specific mRNA expression profiles of three human adult brain regions were generated by next generation sequencing using Illumina GAIIx
Project description:Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of candidate CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery of candidate CREs to target organs in vivo. As a proof-of-concept, we introduce a capture library of ~46,000 constructs, corresponding to ~3,500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as non-human primates and human stem cell-derived organoids.
Project description:Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-Seq, an improved form of the Assay for Transposase-Accessible Chromatin coupled with next-generation sequencing (ATAC-Seq), to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis, and limb development. In addition, we use short- and long-read RNA-Seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We leverage a variety of bioinformatic tools to discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions, and correlate gene expression with accessibility.
Project description:Targeted enrichment-based next-generation sequencing or whole exome sequencing were taken for patients with hypomyelinating leukodystrophies to reveal genetic aetiologies. All genomic DNA used in the experiments was extracted from the peripheral leukocytes. A complete kit was synthetized using the Agilent SureSelect Target Enrichment technique, capturing the coding regions from 104 candidate genes, including their exons and exon-intron boundaries (11,473 probes, 383.065 kbp in total). The following NGS which included equipment and reagents was performed on an Illumina NEXTSEQ500 platform manufactured by Illumina (San Diego, California, USA) using paired-end sequencing of 110 bp. The clean paired-end reads were aligned to the human reference genome build hg19, which was previously annotated using ANNOVAR, in addition to insertion-deletion (indel) and single-nucleotide polymorphism (SNP) calling.