转录组定量与标准化

数据标准化的原因？

不同基因的长度不同
不同批次数据的测序量不同

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ref:Computational methods for transcriptome annotation and quantification using RNA-seq

样本内标准化

RPM/CPM

• RPM: Reads per million mapped reads
• CPM: Count per million mapped reads
  
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RPKM/FPKM

• RPKM: Reads per kilo base per million mapped reads
• FPKM: Fragment per kilo base million mapped reads
  
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在双端测序中：FPKM=RPKM/2

TPM

• TPM: Transcript per million
  
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TPM强行要求基因最终加和相同，高表达基因会将整体的表达量拉高

样本间的标准化

直接计算比例

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• Cj 表示样本j的系数或矫正因子
• Dj表示样本j的总测序量

Quantile

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• Cj 表示样本j的系数或矫正因子
• Dj表示样本j的总测序量
• Q(p)j表示样本j的p分位数

REL(Relative Log Expression)

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TMM(Trimmed Mean of M-value)

基本统计学概念

标准差（Standard Deviation）

• 总体标准差：
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• 样本标准差：
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• 标准误差：
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MetaboAnalyst标准化的方法包括

• Sample Normalization
  • Sample-specific normalization
  • Normalization by sum
  • Normalization by median
  • Normalization by reference sample (PQN)
  • Normalization by a pooled sample from group
  • Normalization by reference feature
  • Quantile normalization
• Data transformation
  • Log transformation
  • Cube root transformation
• Data scaling
  • Mean centering (mean-centered only)
  • Auto scaling (mean-centered and divided by the standard deviation of each variable)
  • Pareto scaling (mean-centered and divided by the square root of the standard deviation of each variable)
  • Range scaling (mean-centered and divided by the range of each variable)

中位数相同

DEqMS蛋白数据差异分析
Use boxplot to check if the samples have medians centered. if not, do median centering.

# Here the data is already median centered, we skip the following step. 
# dat.log = equalMedianNormalization(dat.log)

参考

• R语言——标准化与归一化
• What the FPKM? A review of RNA-Seq expression units
• countToFPKM
• How to perform FPKM on raw reads in R