Hi there,
I am using STAR+cufflink combination to handle the unstranded paired-end RNA-Seq datasets.
- I wonder if there are a set of typical parameters for both STAR and cufflink?
For STAR:
STAR --runThreadN 1 --runMode alignReads --genomeDir index --readFilesIn sample_r1.fq sample_r2.fq --outFileNamePrefix sample_ --outSAMtype BAM SortedByCoordinate --outSAMattributes All --outSAMstrandField intronMotif
For cufflink:
cufflinks -p 1 -G sample.gtf -o sample_clout sample_Aligned.sortedByCoord.out.bam
By running the above two command lines, I encountered a warning message:
"Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided."
Does this warning matter? Is anything gone wrong?
- I noticed from the STAR manual that for unstranded RNA-Seq data, we should give the parameter "--outSAMstrandField intronMotif" to STAR. And I tested the following three scenarios:
(1) without --outSAMattributes without --outSAMstrandField, the error message from Cufflinks: BAM record error: found spliced alignment without XS attribute (1) --outSAMattributes All --outSAMstrandField intronMotif, no error message, I can see the XS attribute in the BAM file. (1) --outSAMattributes Standard --outSAMstrandField intronMotif, no error message, I can see that there is NO XS attribute in the BAM file.
Does this mean using either "--outSAMattributes All" and "--outSAMattributes Standard" will get to the same destination? Does Cufflinks treat them in the same way?
Thanks for your help,
Tom