What methods are used during normalization of RNAseq data in the secondary GeneGlobe Data Analysis Center?

Two methods are available: Average Reference Genes’ Molecular Tags or Total Molecular Tags.

Average Reference Genes’ Molecular Tags Method: This method uses the geNorm method to identify the most stably expressed genes across all of the samples being evaluated from the set of reference genes within the panel. The actual normalization factor is the average number of molecular tags for the genes identified to be stably expressed. Genes that have a stability factor of less than 1.5 are considered to be stably expressed. The genes are ranked by most stable to least stably expressed. For additional details on the calculations, please review the following publication:

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002 Jun 18;3(7):RESEARCH0034. PubMed ID: 12184808.

Total Molecular Tags Method: This method uses the total molecular tag count for a sample to normalize the molecular tag count of every gene. This method assumes equal RNA input and that the genes are expressed at a relatively stable level across the samples being compared. The samples should all be from the same cell or tissue type in order to make accurate comparisons. The software will indicate “OK” if more than half of the molecular tag counts are derived from stably expressed genes. If not, the software will report “Caution.”


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