Next Generation Sequencing Facilitates Quantitative Analysis of basic diet-treated (CON)，lipopolysaccharide-treated(LPS), or lipopolysaccharide+genistein treated broiler chicks (GEN) thymic Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of biological process. The goals of this study are to characterize the effects of genistein on the thymic transcriptome of chicks, when suffering from lipopolysaccharide challenge using RNA-Seq technology Methods: Thymic mRNA profiles of 21-day-old chicks with basic diet and lipopolysaccharide-treated, or lipopolysaccharide+genistein treated chick mice were generated by deep sequencing, in triplicate, 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 Results: Using an optimized data analysis workflow, we mapped about 70 million sequence reads per sample to the chicken genome and identified 25,016 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 6 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. We identified 712 DEGs (367 upregulated and 345 downregulated) between the LPS vs CON group with a |fold change| ＞1 (P ≤0.05, Figure 4 a). There are 1926 DEGs (1014 upregulated and 912 downregulated) in the GEN group compared with the LPS groupAltered expression of 6 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Conclusions: Our research has evidenced a novel finding in chicks that dietary genistein could inhibit the increased percentage of CD3+ T lymphocytes, and CD4+/CD8+ T lymphocyte ratio in the peripheral blood induced by lipopolysaccharide injection. Furthermore, dietary genistein exposure altered the mRNA expression profile and AS signatures in the thymus, and regulated the immune response, along with the improvement of thymus index and apoptotic index after lipopolysaccharide challenge. It would help to understand the role of dietary genistein to the regulation of immune function and provide theoretical support for improving healthy production of poultry. Thymic mRNA profiles of 21-day old basic diet-treated, lipopolysaccharide-treated, or lipopolysaccharide+genistein treated chicks

研究背景与目的：下一代测序（Next-generation sequencing, NGS）彻底革新了生物学过程的系统性分析。本研究旨在利用RNA测序（RNA-Seq）技术，表征脂多糖（Lipopolysaccharide, LPS）刺激条件下，染料木黄酮（genistein）对雏鸡胸腺转录组的调控效应。
方法：本研究通过Illumina GAIIx平台进行三次生物学重复深度测序，获取21日龄基础饮食处理组、脂多糖处理组以及脂多糖+染料木黄酮处理组雏鸡的胸腺mRNA表达谱。对通过质量过滤的测序读段，采用两种方法在转录本异构体水平开展分析：其一为Burrows–Wheeler比对工具（Burrows–Wheeler Aligner, BWA）结合方差分析（ANOVA），其二为TopHat结合Cufflinks。采用TaqMan与SYBR Green荧光定量实验完成qRT-PCR验证。
结果：通过优化的数据分析流程，我们将每个样本约7000万条测序读段比对至鸡参考基因组，并通过TopHat流程鉴定出25016个转录本。RNA测序数据证实了25个已知持家基因的稳定表达，其中6个经qRT-PCR验证。RNA测序数据与qRT-PCR结果在超过四个数量级范围内呈线性相关，拟合优度（R²）为0.8798。在脂多糖组与对照组（CON）之间，我们筛选得到712个差异表达基因（differentially expressed genes, DEGs），其中367个上调、345个下调，筛选标准为|折叠变化|＞1（P≤0.05，图4a）。与脂多糖组相比，染料木黄酮处理组存在1926个差异表达基因，其中1014个上调、912个下调。通过qRT-PCR验证了6个基因的表达变化，证实了RNA测序方法的高灵敏度。对差异表达基因进行层级聚类分析，发现了若干尚未被功能表征的基因，其可能参与视网膜功能调控。
结论：本研究在雏鸡中发现了全新的现象：日粮添加染料木黄酮可抑制脂多糖注射诱导的外周血CD3+ T淋巴细胞百分比升高以及CD4+/CD8+ T淋巴细胞比值异常。此外，日粮染料木黄酮暴露可改变雏鸡胸腺的mRNA表达谱及可变剪接（alternative splicing, AS）特征，调控免疫应答，并改善脂多糖刺激后的胸腺指数与凋亡指数。该研究有助于阐明日粮染料木黄酮对免疫功能的调控机制，可为家禽健康养殖提供理论支撑。
21日龄基础饮食处理组、脂多糖处理组及脂多糖+染料木黄酮处理组雏鸡的胸腺mRNA表达谱