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The following normalization methods will generate different results depending on whether the transformation was performed on samples or on features:
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Note that each task can only perform normalization on samples or features. If you wish to perform both transformations, run two normalization tasks successively. To normalize the data, click on a method from the left panel, then drag and drop the method to the right panel. Add all normalization methods you wish to perform. Alternatively, you can click on the green plus button () on each method to add it. Multiple methods can be added to the right panel and they will be processed in the order they are listed. You can change the order of methods by dragging each method up or down. To remove a method from the Normalization order panel, click the minus button () to the right of the method. Click Finish, when you are done choosing the normalization methods you have chosen.
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Normalization Methods
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- Normalize the reads by the length of feature, it generate reads per kilobase
RPKsf = Xsf / Lf; - Sum up all the RPKsf in a sample
PRKs = ∑Ff=1 FRPKsf - Generate a scaling factor for each sample by normalizing the PRK of the sample to the sum PRK of all the samples
where TR is the total reads across all samples , - Divide raw reads by the scaling factor to get TPM
TXsf = Xsf/Ks
- Normalize the reads by the length of feature, it generate reads per kilobase
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Expression signal
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References
Bolstad BM, Irizarry RA, Astrand M, Speed, TP. A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics. 2003; 19(2): 185-193.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7): 621–628.
- Bullard JH, Purdom E, Hansen KD, Dudoit S. Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics. 2010; 11: 94.
- Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11: R25.
- Dillies MA, Rau A, Aubert J et al. A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Brief Bioinform. 2013; 14(6): 671-83.
- Wagner GP, Kin K, Lynch VJ. Measurement of mRNA abundance using RNA-seq data. Theory Biosci. 2012; 131(4): 281-5.
- Ritchie ME, Phipson B, Wu D et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(15):e97.
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