Changes between Version 58 and Version 59 of SOPs/rna-seq-diff-expressions

Timestamp:

10/16/18 09:35:51 (6 years ago)

Author:

gbell

Comment:

--

SOPs/rna-seq-diff-expressions

@@ -29 +29 @@
   * **Quantification of raw counts**
 
+    * Is your sequencing library stranded or unstranded?  This information is needed to help these tools accurately count features.  Strandedness of some library prep methods:
+      * "TruSeqStrandedPolyA" reads are stranded in the reverse direction (relative to the transcript orientation).
+      * "SMARTerUltra-lowPOLYA-V4" reads are unstranded.
+    * See [[SAMBAMqc]] (and/or look at mapped reads in a genome browser) to determine or verify strandedness
+
     * Currently our favorite tool for this is [[http://bioinf.wehi.edu.au/featureCounts/|featureCounts]], part of the [[http://subread.sourceforge.net/|Subread]] package.
       * featureCounts is much faster than htseq-count, but the details of its counting method is quite different from that of htseq-count, especially for paired-end reads
@@ -53 +58 @@
     * [[http://www-huber.embl.de/users/anders/HTSeq/doc/count.html|htseq-count]] works fine to get counts for each gene, but it's quite slow.
       * Include same GTF file describing gene models as was used for mapping -- but think carefully about what genes should be included (such as long non-coding RNAs, microRNAs, or piRNAs)
-      * Is your sequencing library stranded or unstranded?  This information is needed to help htseq-count accurately count features.  If the library prep method is "TruSeqStrandedPolyA", for example, the reads will be stranded in the reverse direction (relative to the transcript orientation).
-        * See [[SAMBAMqc]] to determine strandedness
       * Carefully choose the best "mode" to handle reads that don't completely map to exactly one gene.
       * Counting can be also performed transcript level but this may be too much information.