GSE116610
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
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 hsr?66 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 hsr? 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 hsr?66 and hsp90GFP larvae, respectively, when compared with similar age wild type larvae, with ~35% (45 genes) being common in hsr?66 and hsp90GFP larvae. Similarly, 309 and 232 genes were significantly up-regulated, compared to similar age wild type larvae, in hsr?66 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 hsr?66 and hsp90GFP genotypes suggest that hsr? transcripts and Hsp83 may be involved in several common pathways. Compared to the relatively small number of genes that were commonly affected in hsr?66 or hsp90GFP individuals, the proportion of genes showing unique down- or up-regulation in hsr?66 hsp90GFP double mutant larvae were much larger (~90% uniquely down- and ~78% uniquely up- regulated). This shows that while substantial reduction of hsr? transcripts or over-expression of Hsp83 affect several genes similarly, the effects become dramatically different and more pervasive when the hsr? transcripts are substantially reduced in a background of elevated levels of Hsp83 so that nearly 1850 genes get uniquely affected. Overall design: Wandering third instar larval RNA profiles of wandering late third instar larvae of WT, hsromega66, Hsp90GFP and Hsp90GFP hsromega66 were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform.