Input data and normalization

The Advanced RNA-Seq plugin requires RNA-Seq input data in the form of Expression Tracks. There is no support for microarray data or the Experiment objects used by the classic Transcriptomics Analysis tools.

Expression Tracks can be created using the RNA-Seq Analysis tool with a choice of possible expression values:

RPKM This is the expression value measured in RPKM [Mortazavi et al., 2008]: RPKM $= \frac{\text{total exon reads}}{\text{mapped reads(millions)} \times \text{exon length (KB)}}$ .
TPM Transcripts Per Million This is computed as $\frac{\text{RPKM}\cdot 10^6}{ \sum \text{RPKM}}$ , where the sum is over the RPKM values of all genes/transcripts (see
Total counts This is all the reads that are mapped to this gene - both reads that map uniquely to the gene or its transcripts and reads that matched to more positions in the reference which were assigned to this gene.
Unique counts This is the number of reads that match uniquely to the gene or its transcripts.

The choice of expression value only affects how Expression Tracks are visualized in the track view. The results from the Advanced RNA-Seq plugin are not affected by this choice, as the most appropriate expression value is automatically selected for the analysis being performed. For detection of differential expression this is the `Total counts' value, and for the other tools this is a normalized and transformed version of the `Total counts' as described below.

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Input data and normalization