I am working with an arodopsis dataset ( two controls and 2 treatment, each with three replicates)and i am performing rna-seq analysis on the dataset. the steps performed were as follows
-building the index using bowtie2
-followed by aligning the reads to a reference using
tophat -o C06_0_NEW -p 20 -G TAIR10.gtf TAIR10.fa At_C06_0.fastq.bz2
-cufflinks to create transcripts for each replicate
cufflinks -o C06_0_cufflinks -g TAIR10.gtf C06_0_out/accepted_hits.bam
-cufmerge t merge the transcripts of all the replicates into one master transcript
cuffmerge -o cuffmerge_out -s Tindex.fa -g TAIR10.gtf gtf_files.txt
-cuffdiff to analyze the deferentially expressed genes
cuffdiff -o C06_cuffdiff_out -b TAIR10.fa -p 20 -L C06_0,C06_1,C06_2 cuffmerge_out/merged.gtf C06_0_out/accepted_hits.bam C06_1_out/accepted_hits.bam C06_2_out/accepted-hits.bam
The problem that i am encountering is that the output of the cuffdiff tool is one of two things
1) a table with no locus annotation for everything but contains vallues.
2) a table with locus annotation but contains no values.
I have tried encorporating the reference annotation into the commands but that seems to have no effect on the output, i have been stuck at this point for the better part of three weeks and i can't seem to find the solution. this is not an assignment or a project for a class, were i am trying to find an easy solution to get the grades, this is my masters thesis work and i could really use some insight, so if anyone could direct me to what i am doing wrong it would be greatly apppreciated.