Changes between Version 3 and Version 4 of SOPs/rna-seq-diff-expressions

Timestamp:

03/25/13 15:26:52 (12 years ago)

Author:

gbell

Comment:

--

SOPs/rna-seq-diff-expressions

@@ -40 +40 @@
          * ''bsub  "samtools sort -n -m 5000000000 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"''
-    * Add a pseudocount (such as 1) to all genes in all samples to
-      * prevent log2 ratios that require dividing by 0
-      * reduce background count noise
-      * reduce problems with statistical methods that don't like 0's
     * 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. 
 
   * **Quantification of FPKM values**
@@ -58 +55 @@
 
   * **Gene filtering**
-    * Remove from the analysis any genes with 0 counts (or counts = pseudocounts).
-    * Remove counts for any genes we want to classify as contaminants such as ribosomal RNAs (if these are included in the GTF gene annotation file).
+    * Remove from the analysis any genes with 0 counts.
+    * Remove counts for any genes we want to classify as contaminants or simply "not interesting" such as ribosomal RNAs (if these are included in the GTF gene annotation file).
 
   * **Normalization**