Project description:Optimal brain function requires that neurons carry out extensive post-transcriptional RNA processing to produce a vast diversity of transcripts. Accurate reconstruction and quantification of highly processed RNA using standard RNA sequencing approaches is challenging due to their short read lengths. Long-read direct RNA sequencing can resolve multiple variations within RNA isoforms by capturing full-length transcripts spanning multiple exon-exon junctions, repetitive regions (e.g. retrotransposons), and intronic structures. Here we produce an isoform-level map of post-transcriptional RNA modifications using Oxford Nanopore Technologies (ONT) long-read sequencing of native RNA strands extracted from heads of Drosophila melanogaster aged to day 10 of adulthood. In addition to identifying 930 transcripts that are not present in the reference transcriptome, we find that almost half of the total detected isoforms have polyadenylated tails in excess of 104 nucleotides and that over 59% of transcripts possessed detectable m6A-modified bases. RNA modifications are present in RNA transcribed from transposable elements, which are important drivers of genetic diversity and relevant to human neurodegenerative diseases, including Alzheimer’s disease and related tauopathies. Applying nanopore direct RNA sequencing to a Drosophila model of tauopathy with known transposable element activation and various types of errors in RNA handling reveals exceptionally diverse RNA processing events in regions that are considered difficult to characterize with traditional short-read sequencing. Taken together, we have uncovered complex transcript structures in adult Drosophila head in a physiological setting and in the context of tauopathy, laying the groundwork for future studies to characterize the diverse tau transcriptome in brain tissue from patients with Alzheimer’s disease and related tauopathies.
Project description:a chromosome-level nuclear genome and organelle genomes of the alpine snow alga Chloromonas typhlos were sequenced and assembled by integrating short- and long-read sequencing and proteogenomic strategy
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:Provide a comprehensive picture of HERV RNA expression through both short and long read sequencing in NCCIT cells to be used in an integrated proteogenomic analysis pipeline
Project description:ChIP-Seq profiles of MSL1, MSL2, MSl3, MOF, MLE, H4K16ac and RNA Polymerase II phosphorlyated on Serine 5 in Drosophila S2 cells MSL1, MSL2, MSL3, MOF, MLE, H4K16ac and RNA Polymerase II phosphorlyated on Serine 5 ChIP in Drosophila S2 cells. 1-3 biological replicates per experiment. Performed in single-read and paired-end read mode.