I am analyzing RNAseq data using cufflinks but I have some confusion regarding the the flow of analysis.
I have RNAseq data for 4 condition say A,B,C and D each having 2 replicate say R1 and R2.
steps which I am following
1. Mapped the reads(using bowtie2) ---> output : 8 aligned sam and bam file(A_R1, A_R2, B_R1 , B_R2.......)
2. cufflink (e.g : cufflink -o A_R1 -G mm10 A_R1) ----> output : 8 transcript.gtf (A_R1.gtf, A_R2.gtf......)
3. cuffmerge (e.g Cuffmerge -o A -g mm10 assembly _GTF_list.txt) . assembly _GTF_list.txt has list of transcript.gtf for each replicate (for e.g A_R1.gtf and A_R2.gtf) ----> output : merged.gtf
Now i have 4 merged.gtf for 4 condition.
First of all I would like to know wheather I am going in right direction or not and secondly I am confused with the use of cuffquant and cuffdiff. I will appreciate any help.
Thanks in advance!