Next-generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing sevenless-GAL4 directed down or up regulated levels of hsromega lncRNAs levels. Next-generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing sevenless-GAL4 directed down or up regulated levels of hsromega lncRNAs levels

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. Overall design: Eye discs mRNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi and sev-GAL4>EP3037 third instar larvae were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform.

研究目的：比较经sev-GAL4驱动下调或上调hsrω长链非编码RNA（lncRNAs）后，三龄幼虫眼盘的转录组变化。
方法：采用Illumina HiSeq2500平台，对sev-GAL4>UAS-GFP、sev-GAL4>UAS-hsrωRNAi、sev-GAL4>EP3037三种基因型的晚期三龄游走幼虫眼盘总RNA进行测序，每个样本设置2次生物学重复；测序采用50bp双端读段，每泳道上样6个样本，每个样本在2个泳道中完成测序，最终每个样本的测序深度约为2000万条读段。将得到的FASTQ格式测序文件通过搭载Bowtie的TopHat比对至果蝇基因组（dm6），利用Cufflinks 2.1.1对每个样本的SAM/BAM格式比对文件进行引导式转录本组装并计算基因计数。随后将比对后的读段使用Cufflinks进行组装，并通过Cuffmerge整合所有样本的转录本，得到跨样本的最终转录组组装结果。为确定不同样本间基因转录本的差异表达情况，采用Cuffdiff 2.1.1进行分析（Trapnell et al., 2012）。
结果：通过优化后的数据分析流程，我们将每个样本约2000万条测序读段比对至果蝇基因组（dm6），并在每个研究的基因型中通过TopHat流程鉴定出15905条转录本。与sev-GAL4>UAS-GFP眼盘对照组相比，经sev-GAL4驱动的hsromega基因RNAi在眼盘中的表达导致大量基因差异表达，其中409个基因下调、100个基因上调。相较于对照组眼盘，在hsrω RNA上调的样本中，有66个基因上调、635个基因下调；其中11个基因通过qRT-PCR得到验证。
结论：对RNA-seq数据的分析显示，在正常背景与激活的Ras表达背景下，hsrω RNA的下调或上调均可对大量转录本产生相似影响。对sev-GAL4>hsrωRNAi与sev-GAL4>EP3037眼盘转录组的比较发现，两种基因型中下调的基因数量均多于上调基因。有趣的是，两种基因型中共有的下调基因达319个、共有上调基因15个，而仅有2个基因在sev-GAL4>UAS-hsrωRNAi与sev-GAL4>EP3037眼盘中呈现表达趋势相反的情况。
整体实验设计：采用Illumina HiSeq2500平台，对sev-GAL4>UAS-GFP、sev-GAL4>UAS-hsrωRNAi及sev-GAL4>EP3037三龄幼虫的晚期游走幼虫眼盘mRNA进行测序，每个样本设置2次生物学重复，以此生成各样本的mRNA表达谱。