1-I obtained the RNAseqV2 raw counts, as another post suggested not to use the RSEM.GENE.NORMALIZED as they still contain irregularity as seen on the diag.boxplot
2- I use different methods of normalization to be able to start the clustering analysis.
3-quantile normalization in the preprocess core package, EDAseq withinlanenormalizaetion function, DESeq rlog using design~1,EDgeR COM and calnormfactor were used and all have different values. I don't know which one to use and if I can use quantile normalization for the normalized RSEM gene counts directly.
4-After clustering for data exploration and obtaining , for example, 3 groups can I renormalize (BASED ON THE NEW GROUPING) and assess differential gene expression. Simply cause the conditions or design are not yet known at the time of initial normalization, and I will depend on clustering to create these groupings.
Your help is greatly appreciated.