Changes between Version 53 and Version 54 of SOPs/mapping

Timestamp:

09/06/17 10:07:53 (8 years ago)

Author:

krichard

Comment:

--

SOPs/mapping

@@ -230 +230 @@
   * '''--outSAMtype <BAM sortingMode> ''' Specifies the type of BAM file to create.  Options: 'BAM Unsorted', 'BAM SortedByCoordinate', 'BAM Unsorted SortedByCoordinate' (to create both unsorted and sorted BAMs)
 
-To improve identification of novel splice junctions, run STAR for a second pass, using junctions found from all samples during the first-pass mapping.  This is especially useful if no GTF file (with annotated junctions) was available for the first-pass mapping.
-
-Sample multiple-sample second-pass command (to run after typical mapping [1st pass]):
-{{{
-bsub STAR --genomeDir /path/to/GenomeDir --readFilesIn /path/to/read1.fq.gz /path/to/read2.fq.gz --sjdbScore 2 --outFileNamePrefix whateverPrefix --runThreadN 8 --readFilesCommand gunzip -c --outSAMtype BAM SortedByCoordinate --sjdbFileChrStartEnd sample1_junctions.tab [sample2_junctions.tab ...]
-}}}
-
-Sample single-sample 2-pass command (includes 1st and 2nd passes):
-{{{
-bsub STAR --genomeDir /path/to/GenomeDir --readFilesIn /path/to/read1.fq.gz /path/to/read2.fq.gz --sjdbScore 2 --outFileNamePrefix whateverPrefix --runThreadN 8 --readFilesCommand gunzip -c --outSAMtype BAM SortedByCoordinate --twopassMode Basic
-}}}
+To improve identification of novel splice junctions, run STAR for a second pass, using junctions found from all samples during the first-pass mapping.  This is especially useful if no GTF file (with annotated junctions) was available for the first-pass mapping. Furthermore, this procedure is recommended for improving sensitivity of variant calling with RNA-seq data.  
+
+'''Index the reference genome for First Pass.'''
+        Create folder, "FirstPass" before running these commands. 
+
+To generate FirstPass genome index files for STAR:
+{{{
+bsub STAR --runMode genomeGenerate --genomeDir /path/to/GenomeDirFirstPass --genomeFastaFiles /path/to/genome/fasta --sjdbGTFfile /path/to/GTF/FileName.gtf --sjdbOverhang 100 --runThreadN 8 
+}}}
+
+To map your reads:
+    '''Run this command within the FirstPass directory'''
+{{{
+bsub STAR --genomeDir /path/to/GenomeDirFirstPass --readFilesIn /path/to/Reads_1.fastq  --outFileNamePrefix whateverPrefix --runThreadN 8
+}}}
+
+'''re-index the reference genome for Second Pass.'''
+        Create folder, "SecondPass" before running these commands. 
+
+To improve sensitivity for variant detection it is recommended to run STAR for a second pass, using junctions found from the first-pass mapping.
+To generate theSecondPass genome index files for STAR:
+{{{
+
+bsub STAR --runMode genomeGenerate --genomeDir /path/to/GenomeDirSecondPass --genomeFastaFiles /path/to/genome/fasta --sjdbFileChrStartEnd /path/to/first/pass/directory/SJ.out.tab --sjdbGTFfile /path/to/GTF/FileName.gtf --sjdbOverhang 100 --runThreadN 8 
+}}}
+
+To map your reads:
+    '''Run this command within the SecondPass directory'''
+{{{
+Input format: fastq ; output format: SAM
+bsub STAR --genomeDir /path/to/GenomeDirSecondPass --readFilesIn /path/to/Reads_1.fastq  --outFileNamePrefix whateverPrefix --runThreadN 8
+}}}
+
 
 == Others ==