Unknown,Transcriptomics,Genomics,Proteomics

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Differential Gene and Transcript Expression Analysis with TopHat and Cufflinks

ABSTRACT: This submission includes the sample data for a protocol covering differential expression analysis with TopHat and Cufflinks. The protocol also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-Seq analysis results. While the procedure assumes basic informatics skills, these tools assume little to no background with RNA-Seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The example data was generated in silico to closely resemble a real experiment in Drosophila melanogaster. First, expression values in cultured S2 cells were calculated for FlyBase 5.2 transcripts. These values were used to generate 3 sequencing replicates for condition "C1", with underlying variability in expression across replicates simulated by fitting a negative binomial model through the real S2 read count data. A second simulated condition "C2" was generated by perturbing expression for 300 randomly selected genes. Genes were perturbed by selecting the most highly expressed isoform and increasing its relative expression by three fold. Three replicates of this condtion were sequenced as above. Simulated sequencing was performed by picking a transcript from the FlyBase transcriptome with equal to its abundance, choosing a fragment length from a normal distribution with mean = 180bp and standard deviation = 20bp, and then choosing a start point for the fragment within the transcript uniformly at random. Total sequencing yield for each replicate was chosen to match that of the real S2 data. Each replicate was mapped to the fly genome with TopHat v 1.3.1 seperately. The replicates were assembled seperately with Cufflinks v 1.1.0. The replicate assemblies were merged with Cuffmerge. This merged assembly was then analysed for differentially expressed and regulated genes with Cuffdiff.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Cole Trapnell

PROVIDER: E-GEOD-32038 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The goal of this study was to compare gene expression in whole embryos to identify transcriptomic changes that result from maternal exposure to predation risk. Methods: Whole embryo mRNA profiles of 3 day post-fertilizationstickleback embrosof mothers exposed to simulated predation risk and control embryos were generated by RNA-sequencing of pooled embryos using Illumina Hiseq2000. The sequence reads that passed quality filters were aligned to the stickleback reference genome and analyzed at the gene level (EdgeR) and at the transcript level (Cufflinks/Cuffdiff). Subsets of embryos were also measured for embryo length and eye diameter, and data were analyzed with a general linear model (SPSS). Results: We mapped ~22 million sequence reads per sample to the stickleback reference genome (BROADS1, Ensembl database version 71.1, Feb 2006) and identified 17440 transcripts with the Tophat workflow. Differential expression analysis using both EdgeR and Cufflinks/Cuffdiff identified 455 transcripts were differentially expressed in embryos of mothers exposed to simulated predation risk as compared to control embryos, with an FDR <0.05 (Cuffdiff) or <0.10 (EdgeR). Gene ontology and pathway analysis (DAVID, IPA) of the differentially expressed gene list revealed enrichment of genes involved in growth, metabolism, neurogenesis, and epigenetics. Embryos of mothers exposed to predation risk had elevated expression of growth and metabolism genes and were also larger than control embryos, suggesting at least some of the genes differentially expressed in this study are involved in the transfer of maternal experience to offspring. Conclusions: Our results suggest that early stickleback embryos respond to maternal exposure to predation risk via changes in gene expression, and a general acceleration of the developmental program. Further study is needed to elucidate the myriad molecular interactions between genes that are differentially-regulated as a result of maternal exposure to predation risk and to understand their relationships to previously-observed maternal effects in this system. Whole embryo mRNA profiles of 3dpf stickleback embryos of mothers exposed to simulated predation risk [E] and control mothers [C] were generated by barcoded, multiplexed high-throughput RNA-sequencing on Illumina Hiseq-2000.

Project description:When C. elegans larvae hatch in the absence of food they persist in a stress resistant, developmentally arrested state (L1 arrest) for weeks or until food becomes available. We characterized growth, mRNA expression, and RNA Polymerase II activity genome-wide during L1 arrest and recovery. RNA Pol II binding data resulting from ChIP-Seq experiments using the Illumina Genome Analyzer are included in this GEO submission. Complementary mRNA expression data from the Affymetrix microarray platform can be found at GEO accession# GSE11055. The goals of the Pol II ChIP-Seq compononent of this project were to use Pol II antibodies 1) to investigate patterns of transcription in developmentally arrested larvae, when mRNA expression levels have reached steady state; 2) to investigate patterns of transcription in immediate response to feeding, when mRNA expression levels change dramatically; and 3) to investigate accumulation of Pol II at promoters during arrest and recovery. We started our ChIP studies with the S2 antibody (Abcam ab5095) since it was raised against a Ser2 phosphorylated peptide from the C-terminal domain of Pol II and should therefore be relatively specific for active, elongating Pol II, and it worked well for the first two goals above. In order to investigate accumulation of Pol II at promoters, we used the S2 antibody and complemeted it with antibody 4H8 (Abcam ab5408), which according to the manafacturer recognizes phosphorylated and non-phosphorylated Pol II, and antibody 8WG16 (Abcam ab817), which binds primarily to non-phosphorylated Pol II but also relatively weakly to phosphorylated Pol II. We were somewhat surprised to find that the results obtained with each of these antibodies were very similar, though upon deeper analysis we discovered relatively subtle differences consistent with the relative specificities of each antibody and existing models regarding phosphorylation state of Pol II and its activity and location. In summary, we find that while Pol II continues transcribing starvation genes, it is M-bM-^@M-^XpausedM-bM-^@M-^Y accumulates on the promoters of growth and development genes during L1 arrest. Consistent with it poising arrested larvae for recovery, pausingpromoter accumulation decreases in response to feeding, while elongation and mRNA levels increase. These results demonstrate that Pol II pausing is widespread in C. elegans and that it is nutritionally controlled during development.These results demonstrate that accumulation of Pol II at promoters of growth and development genes is common in C. elegans and that promoter accumulation anticipates nutritionally controlled gene expression during development. RNA Pol II binding was examined with three different antibodies (S2, 4H8, and 8WG16) during either L1 arrest (starvation) or after 1 hr recovery by feeding. For the S2 antibody two different time points during L1 arrest were examined (6 and 12 hr). 14 total samples are included: 4 independent control samples (Input), a pair biological replicates with the S2 antibody at 6 hr L1 arrest, a pair of biological replicates with the 4H8 antibody at 12 hr L1 arrest and at 1 hr recovery, and singletons for the S2 and 8WG16 antibodies at 12 hr L1 arrest and at 1 hr recovery. "GSE13973_PolII_ChIP-Seq.xls.gz" contains 5 worksheets with the following contents: "geneDensity" includes the number of reads per million mapping to each gene model normalized to gene length. Units are reads per million per kilobase. "TSSdensity" includes the number of reads per million mapping to a 200 bp window spanning the most upstream transcription start site for each gene and normalized by length (200 bp). Units are reads per million per kilobase. "geneEnrichment" includes the fold-enrichment of read density over each gene model relative to input. Where input values were below the median of all input values then the median was used. Units are fold-enrichment. "TSSenrichment" includes the fold-enrichment of read density over the 200 bp TSS window relative to input. Where input values were below the median of all input values then the median was used. Units are fold-enrichment. "5' bias" includes 5' bias calculation for each gene and each antibody in each condition. "GSE13973_CelegansWS190_GeneCoordinates.xls.gz" contains the gene coordinates based on version WS190 of the C. elegans genome.

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Differential Gene and Transcript Expression Analysis with TopHat and Cufflinks

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