SEQanswers Genomatix RNA-Seq workflow
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 02-09-2012, 08:50 AM #1 rebrendi ng   Location: LA Join Date: May 2008 Posts: 78 Genomatix RNA-Seq workflow Hello, has anyone used Genomatix for RNA-Seq analysis? I am particularly interested in the interpretation of the output of the Audic-Claverie algorithm for differential gene expression analysis described here: http://www.genomatix.de/online_help/.../claverie.html Can someone tell me how is calculated the "NE value" for differential expression? How it relates to RPKM? Thank you!
 02-13-2012, 08:13 AM #2 Bernward Junior Member   Location: germany Join Date: Feb 2012 Posts: 1 Hi, The NE is explained at: http://www.genomatix.de/online_help/...lustering.html Normalized expression/enrichment value (NE-value) The NE-value is calculated based on the following formula: NE = c * #readsregion / (#readsmapped * lengthregion) where NE is the normalized expression or enrichment value, #readsregion: the reads (sum of base pairs) falling into either the transcript or the cluster region, #readsmapped: all mapped reads (in base pairs), lengthregion: the transcript or cluster length in base pairs and c a normalization constant set to 10E7. This is quite similar to RPKM. Regarding Audic & Claverie statistics: This was originally designed for the count data in cDNA / Sage libraries. "The Significance of Digital Gene Expression Profiles" Audic & Claverie, 1997. The idea is to calculate the conditional probability to find y counts for your sequence feature (gene, transcript, ChIP region) in your treatment data when you saw x counts in the control for the same sequence feature. However, for NGS data this calculation overestimates the number of significantly deviant features, since it is based on a Poisson model. As discussed here several times the Poisson model doesn't fit well for NGS data. If replicates are available I strongly recommend using DESeq or EdgeR. For ChIP-seq clusters however, you must merge or intersect the clusters from your replicates. The workflow is explained at: http://www.genomatix.de/online_help/...qWorkflow.html Regards, Bernward