I am working with an Arabidopsis 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
) - cuffmerge 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 values.
or
2) a table with locus annotation but contains no values.
I have tried incorporating 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 appreciated.
Thank You
Did you provide the annotated GTF with cuffmerge?
Yes, I just forgot to add the altered command in the question.
The result of adding the annotated gtf file was annotated genes but with no values when run through cuffdiff.
Is that your complete cuffdiff command? If it is, I'd imagine that it's having trouble, because you're telling it to test three sample types in singlet. it should be
blah.gtf condA_rep1.bam,condA_rep2.bam condB_rep1.bam,condB_rep2.bam
... if I remember rightly...