Hi
I am just starting up and getting to know and analyze my data from the RNA-seq. I have 2 biological replicates for 2 different conditions, Pair-end sequenced. The workflow is as follows: I ran FastQgroomer on all my reads, then I used these reads to align to a reference genome using Tophat2. Tophat 2 analysis showed about 80 % alignment with the reference. Then I ran Cufflinks on the accepted hits from Tophat2 with the reference genes and genome. I verfied the cufflinks output files and I can see FPKM values for the two different conditions and replicates. After this i used cuffmerge to merge the transcripts from all the cufflinks outputs. so i merged the four assembled transcripts files from cufflinks( 2 conditions and 1 replicate for each conditions). Then i ran cuffdiff to find differenctial expresssions and so on, using the merged transcript file from cuffmerge and i also used the reference genome. In the cuffdiff output..differential gene/transcript expression tracking files..I see the FPKM is totally zero and i see 'NOTEST' for all the samples.
What does that mean ? I dont think that means that there are no significanlty expressed genes between the samples ! . When i compare the FPKM values from cufflinks, there is big differene in FPKM values for the 2 conditions !
What can be done to solve this ? Please suggest !
Thank you and Best Regards
I am having the same problem and I am trying to use the .gtf annotation file with the merged.gtf file from Cuffmerge on the Cufflinks. Does someone knows how to use both together in a workflow???