Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and miR-211-/- Whole Eye Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS transcriptome profiling (RNA-seq) from whole eye, after removal of the lens and cornea from 1-2 month old miR-211-/- mice and compare it with wt mice Methods: Whole eye (after removal of the lens and cornea) mRNA profiles of 1-2 month old wild-type (WT) and neural miR-211-/-mice were generated by deep sequencing, in multiple biological replicates, five for WT and six for miR-211-/- animals, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays RNA-Seq libraries were prepared from whole eye, after removal of the lens and cornea from miR-211-/- mice. Results: Each library was sequenced using 100 bp paired-end sequencing on the Illumina HiSeq 1000 system. Gene abundances from RNA-Seq data were quantified using RSEM45. Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome. This approach yielded read count values for a total of 38253 mouse genes annotated in GenCode. We only considered genes that had at least 1 count per million in at least five out of 11 samples as expressed, yielding a total of 15590 genes. Next we performed differential gene expression analysis to determine the transcriptional effects of miR-211 deletion. This analysis yielded a total of 63 genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 (Fig. 4). Of these, the expression levels of 61 genes were significantly decreased upon miR-211 deletion, while only 2 genes were upregulated. Conclusions: Our study represents the first detailed analysis of whole eye transcriptomes, with biologic replicates, generated by RNA-seq technology on miR-211-/-. Whole eye (after removal of the lens and cornea) mRNA profiles of 1-2 month old wild-type (WT) and neural miR-211-/-mice were generated by deep sequencing, in multiple biological replicates, five for WT and six for miR-211-/- animals, using Illumina GAIIx.研究目的：下一代测序（Next-generation sequencing, NGS）已革命性地变革了系统级细胞通路分析。本研究旨在对比1-2月龄miR-211基因敲除（miR-211-/-）小鼠去除晶状体和角膜后的全眼组织的NGS转录组测序（RNA-seq）结果，并与野生型（WT）小鼠进行对照比较。 实验方法：采用Illumina GAIIx测序平台，对1-2月龄野生型（WT）及神经特异性miR-211基因敲除（miR-211-/-）小鼠去除晶状体和角膜后的全眼组织开展mRNA深度测序，设置多个生物学重复：野生型组5例，miR-211-/-组6例。通过质量过滤的测序读段，采用两种分析方法在转录本异构体层面进行数据分析：一是Burrows-Wheeler比对工具（Burrows-Wheeler Aligner, BWA）结合方差分析（ANOVA），二是TopHat结合Cufflinks。采用TaqMan与SYBR Green试剂盒进行qRT-PCR验证。RNA-seq文库均由miR-211-/-小鼠去除晶状体和角膜后的全眼组织制备。 实验结果：每个文库均通过Illumina HiSeq 1000系统进行100 bp双端测序。利用RSEM45对RNA-seq数据中的基因表达丰度进行定量。通过优化的数据分析流程，我们将每个样本约3000万条测序读段比对至小鼠参考基因组。该分析共得到GenCode注释的38253个小鼠基因的读段计数。我们仅将在11个样本中至少5个样本的每百万读段计数≥1的基因认定为表达基因，最终筛选得到15590个表达基因。随后开展差异基因表达分析，以明确miR-211敲除对转录组的调控影响。该分析共得到63个错误发现率（False Discovery Rate, FDR）<0.1的差异表达基因（图4）。其中，61个基因在miR-211敲除后表达量显著下调，仅2个基因表达上调。 研究结论：本研究首次采用RNA-seq技术对带有生物学重复的miR-211-/-小鼠全眼组织（去除晶状体和角膜）进行了详细的转录组分析。 本研究采用Illumina GAIIx测序平台，对1-2月龄野生型（WT）及神经特异性miR-211基因敲除（miR-211-/-）小鼠去除晶状体和角膜后的全眼组织进行mRNA深度测序，设置5个野生型生物学重复与6个miR-211-/-生物学重复。