Dear all,
I am using cufflinks in Galaxy, but I faced with the problem with FPKMs.
When I use gtf file (hg19: shared data > data libraries > iGenomes )and reference genome for human available in Galaxy(hg19: a tophat option), the cufflinks output gives me genes ID, but most FPKMs are zero.
Can you help me find out the problem?
How can I choose a compatible reference genome for that GTF file?
Thank you in advance
Nazanin
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nazaninhoseinkhan
Hi Nazanin,
can you check the chromosome names in the GTF and the bam file? Do they start with a chr in the GTF and without in the bam -- or vice versa? Is the alignment performed on HG19 annotation?
Cheers,
Michael
Thanks for your response.
Both hg19 gtf and cufflinks output (assembled transcrips) start with chr.
When I run tophat I also use hg19 reference genome.
best
Nazanin
It's not surprisingly that some FPKMs are 0 (depending on your total number of reads and tissue).
-Edited to replace 'a lot of' by 'some'-
Right, edited my response to "some". I guess subjective quantitative terms can be misinterpreted easily ;)
You mean that if FPKM of "some" genes are zero, is OK, due to total number of reads and under studied tissue?
Thanks for your comment.
Yes,your right. The file has 4082 zero out of 65536( or may be more, because my excel cannot show more).
best
Nazanin
That looks like nothing to worry about. These genes are either lowly expressed (and therefore not sequenced) or just tissue-specific.
If you can, avoid Excel as much as possible in bioinformatics analysis. Next to automated formatting (changing floats or gene names into date format), you may experience differences due to floating point arithmetics.
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