Changes between Version 14 and Version 15 of SOPs/rna-seq-diff-expressions

Timestamp:

01/15/14 14:23:08 (11 years ago)

Author:

gbell

Comment:

--

SOPs/rna-seq-diff-expressions

@@ -39 +39 @@
          * ''bsub  "samtools sort -n -m 5G accepted_hits.bam accepted_hitsSortedByname"''
          * To request a certain amount of memory and a specific node use ''bsub  -R "rusage[mem=50000]" -m NodeName '' 
-         * ''bsub  "samtools sort -n -m 5G accepted_hits.bam accepted_hitsSortedByname"''
-         * ''bsub  "samtools view accepted_hitsSortedByname.bam | htseq-count -m intersection-strict --stranded=no - gene_models.gtf >| gene_model.counts.txt"''
     * Remove the rows at the bottom with descriptions like no_feature, ambiguous, etc.
     * For custom analyses, you can add a pseudocount (such as 1) to all genes in all samples to prevent log2 ratios that require dividing by 0 and reduce background count noise -- BUT be aware that some statistical methods (like DESeq) require raw input values without any pseudocounts or normalization. 
@@ -69 +67 @@
        * Scaling so total number of counts is equal to mean number of mapped reads across all samples
        * Effective library size (a more complex, but probably more valid method included in programs such as edgeR and DESeq)
-     * As with microarray normalization, be aware of the assumptions of each method and choose the method(s) which are most valid with your experiment
+     * '''Differential expression statistics packages can output a matrix of normalized counts (typically using the method recommended for the accompanying statistics), so typically no additional normalization needs to be done''' (unless one want to perform further normalization, such as using gene length).
+     * As with microarray normalization, be aware of the assumptions of each method and choose the method(s) which are most valid with your experiment.
      * Origin of recommendation for upper-quartile normalization: [[http://www.ncbi.nlm.nih.gov/pubmed/20167110|Bullard et al., 2010]]
      * See [[http://jura.wi.mit.edu/bio/scripts/R/normalize_DGE_matrix.R|normalize_DGE_matrix.R]] for a command-line script for count normalization