Changes between Version 29 and Version 30 of SOPs/rna-seq-diff-expressions

Timestamp:

06/01/16 10:45:32 (9 years ago)

Author:

gbell

Comment:

--

SOPs/rna-seq-diff-expressions

@@ -30 +30 @@
     * Typically we use [[http://www-huber.embl.de/users/anders/HTSeq/doc/count.html|htseq-count]] to get counts for each gene
       * 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)
-      * 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
+      * Is your sequencing library stranded or unstranded?  This information is needed to help htseq-count accurately count features.
+      * 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.
       * htseq-count can accept SAM or BAM (-f option), for example
-         * ''htseq-count -f bam -m intersection-strict --stranded=no accepted_hits.bam gene_models.gtf >| gene_model.counts.txt''
-         * Note that htseq-count assumes that your reads are strand-specific, so if not, include "--stranded=no" (or half of your reads won't be counted).     
+         * ''htseq-count -f bam -m intersection-strict --stranded=reverse accepted_hits.sortedByName.bam gene_models.gtf >| gene_model.counts.txt''
+         * Note that htseq-count assumes that your reads are strand-specific; default is --stranded=yes
+         * If your reads are not stranded, use "--stranded=no" (or half of your reads won't be counted).  If they're stranded and in the opposite direction from the transcript, use "--stranded=reverse".
          * Note the "-" can be used for input from standard input (stdin)
-         * For paired-end reads the sam file has to be sorted by read name, or coordinate, eg. ''bsub  "samtools sort -n -o accepted_hits.sortedByName.bam -m 5G -O bam -T temp accepted_hits.bam"''
+         * For paired-end reads the sam file has to be sorted by read name: ''bsub  "samtools sort -n -o accepted_hits.sortedByName.bam -m 5G -O bam -T temp accepted_hits.bam"''
          * To request a certain amount of memory and a specific node use ''bsub  -R "rusage[mem=50000]" -m NodeName '' 
          * Remove the rows at the bottom with descriptions like no_feature, ambiguous, etc.