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Next-generation sequence analysis for transcriptome of Drosophila melanogaster whole third instar larvae of wild type (wt), hsromega66 mutant (near null allele), Hsp90GFP (over-expressing Hsp83) and hsromega66 Hsp90GFP recombinants

ABSTRACT: Purpose: To compare transcriptomic changes in 3rd instar larvae due to near-absence of hsromega gene transcripts, or over-expression of Hsp83, or both in combination. Method: Third instar larval total RNA profiles of WT, hsromega66, Hsp90GFP and Hsp90GFP hsromega66 were generated by sequencing, in duplicate, on Illumina Hiseq2500 platform using 50bp pair-end reads, 8 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. Compared to wild type, loss of hsromega gene transcripts due to hsrɷ66 resulted in differential expression of many genes, with128 genes being down-regulated and 309 up-regulated, while that due to over expression of Hsp83 in Hsp90GFP resulted in 154 genes down regulated and 232 up-regulated. Compared to control larvae, in case of up regulation of Hsp83 and simultaneous near absence of hsrɷ transcripts, 1157 gene were up-regulated with 842 showing unique change in only this genotype but not in the former two genotypes, and 1120 were down-regulated with 1005 lowered uniquely in these individuals. Some of these (9 genes) were validated using qRT-PCR. Conclusion: Analysis of RNA-seq data revealed that 128 and 154 genes were significantly down-regulated in hsrɷ66 and hsp90GFP larvae, respectively, when compared with similar age wild type larvae, with ~35% (45 genes) being common in hsrɷ66 and hsp90GFP larvae. Similarly, 309 and 232 genes were significantly up-regulated, compared to similar age wild type larvae, in hsrɷ66 and hsp90GFP larvae, respectively. In this case also ~37% (113) were commonly up-regulated in both these genotypes. Such common effects on a significant proportion of genes in hsrɷ66 and hsp90GFP genotypes suggest that hsrɷ transcripts and Hsp83 may be involved in several common pathways. Compared to the relatively small number of genes that were commonly affected in hsrɷ66 or hsp90GFP individuals, the proportion of genes showing unique down- or up-regulation in hsrɷ66 hsp90GFP double mutant larvae were much larger (~90% uniquely down- and ~78% uniquely up- regulated). This shows that while substantial reduction of hsrω transcripts or over-expression of Hsp83 affect several genes similarly, the effects become dramatically different and more pervasive when the hsrω transcripts are substantially reduced in a background of elevated levels of Hsp83 so that nearly 1850 genes get uniquely affected.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE116610 | GEO | 2018/12/31

REPOSITORIES: GEO

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Project description:Purpose: To compare transcriptomic changes in 3rd instar larval eye discs following sev-GAL4 driven down- or up- regulation of hsrω lncRNAs. Method: Eye disc total RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037 were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 6 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of RNAi for hsromega gene transcripts in eye discs resulted in differential expression of many genes, with 409 genes being down-regulated and 100 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to control eye discs in case of up regulation of hsrω RNA, 66 gene were up-regulated while 635 were down-regulated. Some of these (11 genes) were validated using qRT-PCR. Conclusion: Analysis of RNA-seq data revealed that a large proportion of transcripts were indeed similarly affected by down- or up-regulation of hsrω RNA in normal as well as activated Ras expression background. A comparison of transcriptomes of sev-GAL4>hsrωRNAi and sev-GAL4>EP3037 eye discs revealed that in each case the number of genes down regulated was more than those up-regulated. Interestingly, while 319 genes were commonly down regulated and 15 were commonly up-regulated in the two genotypes, only 2 genes showed opposing trends between sev-GAL4>UAS-hsrωRNAi and sev-GAL4>EP3037 eye discs.

Project description:Purpose: Feeding larvae of fly models of polyQ and other neurodegenerative disorders on AR or RS supplemented food substantially suppressed neurodegeneration. With a view to gain further insight into the suppression of polyQ pathology by AR or RS, we expressed the UAS-127Q transgene using the predominantly eye-specific GMR-GAL4 driver and examined the transcriptomic changes in wild type and 127Q-expressing eye discs following feeding on AR or RS. In order to find out reason underlying the suppression of neurodegeneration by feeding of AR and RS, we checked difference in gene expression between eye discs expressing GMR-GAL4>UAS-127Q and wild type. Method: Eye disc RNA profiles of wandering late third instar larvae of GMR-GAL4>UAS-127Q and wild type larvae, reared on normal food or food supplemented with AR or RS since after hatching, were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 51bp pair-end reads, 12 samples per lane and each sample run across 2 lanes of flowcell (Flowcell ID: C7K52ACXX).This resulted in a sequencing depth of 19-25 million reads. The resulting sequencing fastq files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to merge with Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 19-25 million sequence reads per sample to the Drosophila melanogaster genome (dm6) and identified 15,904 transcripts with cufflink workflow in each genotypes studied. The GMR-GAL4 driven UAS-127Q expression in eye discs reared on AR supplemented food resulted in 259 differential expression, with 147 genes being down-regulated and 112 up-regulated, when compared with those reared on normal food, however, when larvae from same genotype reared on RS supplemented food resulted in 1641 DEGs, with 846 genes being down-regulated and 795 up-regulated compared with larvae reared on normal food. In eye disc of wild type larvae reared on AR supplemented food resulted in 680 DEGs, with 470 genes being down-regulated and 205 up-regulated compared with those reared on normal food. Larvae from wild type reared on RS supplemented food resulted in 427 DEGs, with 264 genes being down-regulated and 163 up-regulated compared with those reared on normal food. Compared to only GMR-GAL4>UAS-127Q and wild type larval eye discs reared on normal food, total of 1691, with 799 gene were up-regulated while 892 were down-regulated. Conclusion: The present results not only re-inforce earlier findings from our lab that the Ayurvedic Amalaki Rasayana and Rasa-Sindoor are potent suppressors of neurodegeneration but also provide direct evidence that these general health-promoting formulations significantly alter expression of many genes in protein-aggregation-clearance pathways and thus eliminate the cause of proteinopathy so that cell death is inhibited while other normal cellular activities continue. These formulations, which have been used in Ayurvedic practice for thousands of years as health-promoting as well as curative supplements, do not have any side-effects. Therefore, we believe that these Ayurvedic formulations have a good potential to ameliorate the sufferings of polyQ and other neurodegenerative disorders.

Project description:Purpose: While studying possible interaction between ras and hsrω genes we found that down- or up- regulation hsrω RNA levels results in further enhancement in activity hyperactive Ras protein in eye discs of Drosophila melanogaster. In order to find out reason underlying this phenomena we checked difference in gene expression between eye discs expressing activated Ras along with alterations in hsrω RNA levels and those expressing activated Ras alone. Method: Eye disc RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037, sev-GAL4>UAS-RasV12, sev-GAL4>UAS-hsrωRNAi UAS-RasV12 and sev-GAL4>EP3037 UAS-RasV12 were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 12 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of activated Ras in eye discs resulted in differential expression of many genes, with 374 genes being down-regulated and 138 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to only Ras expressing eye discs in case of down regulation of hsrω RNA in activated Ras background 148 gene were up-regulated while 434 were down-regulated. In eye discs expressing activated Ras alongwith over expression of hsrω RNA, 192 genes were up-regulated and 470 down-regulated compared to only activated Ras expressing eye discs. Some of these (11 genes) were validated using qRT-PCR. Conclusion: In activated Ras expressing eye discs, besides the expected increase in levels of transcripts of genes involved in cell growth, proliferation and differentiation, transcripts of many genes involved in RNA biosynthesis, metabolism and processing were up-regulated when compared with sev-GAL4>UAS-GFP eye discs. Levels of transcripts of none of the known members of Ras/Raf/MAPK signaling pathway were found to be significantly affected by changes in hsrω transcript levels in activated Ras expression background. Interestingly, several sn/snoRNAs along with a scaRNAs and some members of the RNA processing machinery were differentially expressed in activated Ras background and were further affected when the hsrω RNA levels were down-or up-regulated. In addition, while down-regulation of hsrω activity resulted in up-regulation of a few positive modulators of Ras signaling pathway, up-regulation of these transcripts caused down-regulation of the negative regulators of Ras/Raf/MAPK pathway, and thus resulting in increase in Ras activity in either cases.

Project description:Purpose: To compare transcriptomic changes between 3rd instar wild type larvae and those with highly down regulated hsrω lncRNAs levels Method: Whole third instar larval total RNA profiles of +/+ and +/+;hsromega[66] homozygous were generated by sequencing, in duplicate, on Illumina Hiseq2500 platform using 100bp pair-end reads, which resulted in a sequencing depth of ~20 million reads. The resulting FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. Cuffdiff 2.1.1 was used (Trapnell et al., 2012) to ascertain differential expression of gene transcripts between different samples, . Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 21,529 transcripts with TopHat workflow in each genotype studied, with 20,956 having baseline expression. 134 genes were up regulated and 74 downregulated in +/+;hsromega[66] compared to +/+ whereas 201 expressed only in the wild type sample and 164 only in hsromega[66] mutant Conclusion: Analysis of RNA-seq data revealed that a large proportion of the transcripts affected in larvae with loss of hsromega lncRNA transcripts belonged to innate immune response pathway, mainly those in defense against Gram positive bacteria. Many of these genes are part of proteolysis pathway having endopeptidase activity with predominence of serine-threonine endopeptidases. Kegg pathway analysis revealed that metabolism, especially that of Drugs, was indicated to be affected. Genes showing differential expression belonged to ascorbate and aldarate metabolism,retinol metabolism, pentose and glucoronate interconversion, metabolism of xenobiotics by cytochrome P450

Project description:Purpose: This study aimed to identify differentially expressed genes and transcripts in zebrafish embryos and larvae following benzo[a]pyrene (BaP) exposure. Methods: Adult zebrafish (2 males × 4 females, N=6 replicate tanks for each treatment) were acclimated for 7 days in an 818 Low Temp Illuminated Incubator (Precision Scientific, Chennai, India) at 28.5°C. Next, adult fish were waterborne exposed to control or 50 μg/L (ppb) BaP for 7 days; ethanol was used as vehicle solvent, and final ethanol concentration was 0.1 mL/L (100 ppm) in all treatment groups. This dose of ethanol is not teratogenic to zebrafish. Water was changed and/or re-dosed daily. From day 7 to 11 of the parental exposure, eggs were collected, counted, and raised in normal conditions (control) or continuously exposed to 50 μg/L BaP until 3.3 and 96 hours post fertilization (hpf). At 3.3 or 96 hpf, embryos (200/pool) or larvae (10/pool) were collected and pooled. Total RNA was isolated for transcriptomic RNA sequencing with Illumina HiSeq2000 (2X100bp). RNA-seq reads were uploaded to the galaxy platform https://main.g2.bx.psu.edu/. RNA-seq reads were trimmed, filtered, and aligned to the zebrafish genome (Danio_rerio.Zv9.68) with the Tophat for Illumina tool. Counting and annotation of RNA-seq reads were performed with Partek Genomics Suite version 6.11. Refseq Transcripts (2013-04-10) and Ensembl Transcripts release 70 databases were used for gene and transcript annotation. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level. Results: Differential expression analysis with EdgeR revealed that gene expression was vastly different between 3.3 hpf zebrafish embryos and 96 hpf larvae. Using Refseq annotation, we found that 10644 out of 13950 transcribed zebrafish genes were differentially expressed between the two developmental time-points, with 5961 up-regulated genes and 4683 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. Similarly, using Ensembl annotation, 16529 out of 19886 transcribed zebrafish genes were differentially expressed, with 9318 up-regulated genes and 7211 down-regulated genes in 96 hpf larvae compared with 3.3 hpf embryos. In 3.3 hpf embryos, four genes and seven transcripts were differentially expressed after BaP exposure. In 96 hpf larvae, 447 and 484 zebrafish genes were significantly up- and down-regulated, respectively, by BaP exposure. Conclusions: Parental and developmental BaP exposure caused gene expression changes in zebrafish embryos and larvae.

Dataset Information

Next-generation sequence analysis for transcriptome of Drosophila melanogaster whole third instar larvae of wild type (wt), hsromega66 mutant (near null allele), Hsp90GFP (over-expressing Hsp83) and hsromega66 Hsp90GFP recombinants

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