Project description:High-throughput sequencing of Drosophila melanogaster small RNAs. total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH Keywords: High-throughput solexa sequencing Small RNAs were sequenced from D. melanogaster female head. Raw sequences were clipped by 3' linker sequences recognition, and select clipped sequences longer than 18 nt Quality scores in the supplementary file for GSM240749 are undefined. Quality of the bases assessed by (1) identifying for the sequenced linker, which is a known sequence, and (2) mapping the clipped sequence to the genome and taking only perfect hits.
Project description:The 3' ends of most Drosophila melanogaster genes are poorly annotated or are determined by only a single EST or cDNA clone. To enhance the annotation of poly(A) site use in Drosophila, we performed deep sequencing on RNA isolated from 29 dissected tissues using an approach designed to enrich for poly(A) spanning reads. From these experiments, we identified 1.4 million poly(A) spanning reads leading to the identification of many new poly(A) sites and the identification of many tissue-specific poly(A) sites. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf RNA from 29 dissected Drosophila melanogaster tissues (in duplicate) were used to prepare polyA enriched RNA-Seq libraries. Briefly, total RNA was poly(A) selected, fragmented, and ligated to 5' and 3' RNA linkers. These libraries were amplified using Illumina paired-end primers, and subsequently reamplified using a 3' primer complementary to the 3' adapter but containing 6 Ts at the 3' end. The libraries were also multiplexed and up to 12 samples mixed per lane and sequenced on an Illumina GAIIx using paired-end 76 bp reads, or an illumina HiSeq 2000 using paired-end 100 bp reads. All reads were mapped to the Drosophila melanogaster genome to identify unmapped reads. Unmapped reads containing at least 10 A residues at the 3' end were identified, the terminal A residues trimmed, realigned to the genome to identify uniquely mapped reads. Such reads were identified as polyA spanning reads
Project description:The Y chromosome and other heterochromatic regions present special challenges for genome sequencing and for the annotation of genes. Here we describe two new genes (ARY and WDY) on the Drosophila melanogaster Y, bringing its number of known single-copy genes to 12. WDY may correspond to the fertility factor kl-1.
Project description:Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
Project description:In the past decade, genome-sequencing projects have yielded a great amount of information on DNA sequences in several organisms. The release of the Drosophila melanogaster heterochromatin sequence by the Drosophila Heterochromatin Genome Project (DHGP) has greatly facilitated studies of mapping, molecular organization, and function of genes located in pericentromeric heterochromatin. Surprisingly, genome annotation has predicted at least 450 heterochromatic gene models, a figure 10-fold above that defined by genetic analysis. To gain further insight into the locations and functions of D. melanogaster heterochromatic genes and genome organization, we have FISH mapped 41 gene models relative to the stained bands of mitotic chromosomes and the proximal divisions of polytene chromosomes. These genes are contained in eight large scaffolds, which together account for approximately 1.4 Mb of heterochromatic DNA sequence. Moreover, developmental Northern analysis showed that the expression of 15 heterochromatic gene models tested is similar to that of the vital heterochromatic gene Nipped-A, in that it is not limited to specific stages, but is present throughout all development, despite its location in a supposedly "silent" region of the genome. This result is consistent with the idea that genes resident in heterochromatin can encode essential functions.
Project description:Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. DOI: http://dx.doi.org/10.7554/eLife.01179.001.
Project description:Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p < 0.05). Some eggs displayed abnormal chorionic appendages, some larvae were large and red, and some adult flies showed wing abnormalities. Abnormal wing phenotypes of D. melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in post-transcriptional regulation of metazoan development, homeostasis and disease. The full set of miRNAs is not known for any species and it is believed that many await discovery. The recent assembly of 15 insect genomes has provided the opportunity to identify novel miRNAs in the fruit fly, Drosophila melanogaster. We have performed a computational screen for novel microRNAs in Drosophila melanogaster by searching for phylogenetically conserved putative pre-miRNA structures. The ability of predicted novel miRNA precursors to be processed to produce miRNAs was experimentally verified in S2 cells and in several cases their endogenous expression at was validated by Northern blots. After experimental validation, the predictions were cross-checked with reference to a newly released set of small RNA sequences. Combining both datasets allowed us to identify 53 novel miRNA loci in the fruit fly genome 22 of which we had predicted computationally. This significantly expands the set of known miRNAs in Drosophila melanogaster. Most novel miRNAs contain unique seed sequences not found in other Drosophila miRNAs and are therefore expected to regulate novel sets of target genes. This data provides the basis for future genetic analysis of miRNA function and will aid the discovery of orthologous sequences in other species.
Project description:Here, we report genome assemblies for three strains of Wolbachia pipientis, assembled from unenriched, unfiltered long-read shotgun sequencing data of geographically distinct strains of Drosophila melanogaster Our simple methodology can be applied to long-read data sets of other Wolbachia-infected species with limited Wolbachia-host lateral gene transfers to produce complete assemblies for this important model symbiont.
Project description:To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.