2019/07/16_NGS-Data-Analysis-Course_8_RNA-Seq实验设计

Learning Objectives:

• Describe the importance of replicates for RNA-seq differential expression experiments
• Explain the relationship between the number of biological replicates, sequencing depth and the differentially expressed genes identified
• Demonstrate how to design an RNA-seq experiment that avoids confounding and batch effects

Understanding the steps in the experimental process of RNA extraction and preparation of RNA-Seq libraries ( these two parts information are located in my computer D/meeting/ NGS) is helpful for designing an RNA-Seq experiment, but there are special considerations that should be highlighted that can greatly affect the quality of a differential expression analysis.

These important considerations include:

1. Number and type of replicates 重复的数量和类型
2. Avoiding confounding 避免混淆
3. Addressing batch effects 批次效应

#replicates

Experimental replicates can be performed as technical replicates or biological replicates.

• Technical replicates: use the same biological sample to repeat the technical or experimental steps in order to accurately measure technical variation and remove it during analysis. 同一个样品重复三次，避免技术误差

• Biological replicates use different biological samples of the same condition to measure the biological variation between samples. 三个不同的样品做三次实验在相同的条件下。

如果是老鼠样品，那很容易地获得不同的biological replicates， 但是对细胞系来说获得不同的biological replicates 是有困难的。 https://paasp.net/accurate-design-of-in-vitro-experiments-why-does-it-matter/  这篇文章中给了我们怎么设计细胞系replicates样品。

with the current RNA-Seq technologies, technical variation is much lower than biological variation and technical replicates are unneccessary. In contrast, biological replicates are absolutely essential. For differential expression analysis, the more biological replicates, the better the estimates of biological variation and the more precise our estimates of the mean expression levels. This leads to more accurate modeling of our data and the identification of more differentially expressed genes. 

生物复制越多，对生物变异的估计就越好，我们对平均表达水平的估计就越精确。这将导致我们对数据的更精确建模，并识别出更多差异表达的基因。

Note that an increase in the number of replicates tends to return more DE genes than increasing the sequencing depth.增加模板的复制数量能得到更多的差异基因。Therefore, generally, more replicates are better than higher sequencing depth, with the caveat that higher depth is required for detection of lowly expressed DE genes and for performing isoform-level differential expression.  因此，一般来说，更多的复制比更高的测序深度更好，但需要更高的深度来检测低表达的de基因和执行亚型水平的差异表达。