Project description:Identifying novel proteins involved in iron metabolism using Drosophila Melanogaster larvae.Hsp22 and Hsp70 identified as responsive genes to iron manipulation in an RNA-seq study.
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:This is a dataset which comprises the following two different kinds of genomic data in Drosophila species: First, triplicate ChIP-seq data of CTCF (CCCTC binding factor) binding profiles in each of the four closely related Drosophila species : Drosophila melanogaster, Drosophila simulans, Drosophila yakuba and Drosophila pseudoobscura at white pre pupa stage; Second, triplicate RNA-seq data of white pre pupa whole animals of three Drosophila species: Drosophila melanogaster, Drosophila simulans and Drosophila yakub. The binding site/region/peaks are called using a modified method of QuEST( please see details in our related publication). The sequence read counts and RPKM values are calculated following the method in Mortazavi et al 2008 Nature Methods paper. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
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:Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.
Project description:Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.
2023-06-06 | GSE213984 | GEO
Project description:Detection and identification of transposable element transcripts using Long Read RNA-seq in Drosophila germline tissues
Project description:We used single-embryo metabolomics to characterize early developmental metabolism in Drosophila. We employed a multi-omics approach where samples were collected, homogenized in 80% methanol and the soluble fraction recover to perform targeted metabolomic whle RNA-seq was performed on the insoluble fraction to accurately stage each embryo. Then, this RNA-based staging was used to place single embryo metabolomes across the developmental trajectory. Thus, we are able to construct a highly detailed metabolomic map of embryonic development. Importantly, we validated our single-embryo metabolomics results in pools of 10 embryos. The data provide a continuous timeline of metabolite levels (and gene expression) during early development (0-3 hours) in Drosophila melanogaster. We used two genetically different lines from the Drosophila Genetic Reference Panel (DGRP) with known genetic variations in our crosses (males/DGRP_352, females/DGRP_737). RNA-seq data related to this dataset can be accessed at GEO under accession number GSE263568.