RNA-seq analysis of trait-associated genes (TAGs) in somatic cells from Holstein’s milk

Background : Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, few studies have attempted to characterize the milk synthesizing genes associated with milk yielding traits. In addition, no previous attempts have been made to detect quantitative traits such as milk production related traits, associated genes on RNA-seq experiment with several biological replicates. Research in this field is necessary as bovine milk is a primary source of nutrition for humans. Results : We investigated advantages of using linear models with continuous response variables over converted group variables in a Holsteins’ milk. Suggested methods were more suitable in detection of significant genes in our analysis; the proportion of false discoveries to total significant genes was observed to be much lower compared to the precedent approaches we employed. These were observed through mock comparison studies and quantitative real time PCR (qRT-PCR). Conclusion : Several milk production related genes and pathways were identified from the suggested methods. Given the current trend in RNA-seq pricing, we expect our methods to be successfully applied in various RNA-seq studies with numerous biological replicates that handle continuous response traits. Overall design: We generated 21 RNA-seq samples (biological replicates) from the RNA samples were extracted from the somatic cells of Holsteins’milk.

背景：尽管牛乳（bovine milk）的化学、物理及营养特性已得到广泛研究，但鲜有研究尝试解析与产奶性状相关的乳合成基因。此外，目前尚无研究通过设置多生物学重复的RNA测序（RNA-seq）实验，来筛选与产奶相关性状等数量性状的关联基因。鉴于牛乳是人类的核心营养来源之一，该领域的研究具备重要现实意义。结果：本研究以荷斯坦奶牛（Holstein）牛乳为研究材料，对比了带连续响应变量的线性模型与转换后分组变量的分析效果。结果表明，所提出的分析方法更适于本研究中显著基因的筛选；相较于本研究采用的既往方法，显著基因集合中的假阳性发现占比显著更低。上述结论通过模拟对照实验与实时荧光定量PCR（quantitative real time PCR，qRT-PCR）得以验证。结论：通过所提出的分析方法，本研究筛选出多个与产奶相关的基因及生物学通路。结合当前RNA测序（RNA-seq）的成本趋势，我们预期该方法可成功应用于各类设置多生物学重复、用于分析连续响应性状的RNA测序研究。实验设计概况：本研究从荷斯坦奶牛乳体细胞中提取RNA，并构建了21个RNA测序（RNA-seq）样本（即生物学重复样本）。