Hello again. I am new to bioninformatics and i need some guidance. I am trying to do an RNA seq analysis but the output seems a bit wrong. I got a type of cells for example h at 0,3 and 6 hour, and i got 2 replicates. And i want to compare 0h to 3h and 0h to 6h. So my sampels are: h0b,h0c,h3b,h3c,h6b,h6c(each .fastq, for example h0b.fastq is about 5.8g). I got the gtf for hg19 from table browser in ucsc(Refseq genes). And my pipeline is :
- tophat2 --library-type fr-unstranded -g 1 -G ...hg19.gtf ....bowtie2index/hg19genome h0b.fastq
- cufflinks -g ...hg19.gtf -o ./cufflinks ...acceptedhits.bam
- cuffmerge -g ...hg19.gtf h0-3.txt
- cuffdiff -L h0,h3 ...h0-3merged.gtf h0bacceptedhits.bam,h0cacceptedhits.bam h3bacceptedhits.bam,h3cacceptedhits.bam
I dont specify -p because i have seen many bugs in case i used it. Small in size outputs. And i say a bit wrong output because of the difference of results with a colleague who ran this process in usegalaxy ( but i am trying to learn how to use terminal ) and the gene_exp.diff ( open with excel ) seems a bit off.
Do you see any msitakes i made ? Could i clarify something better for you? Because i am pretty sure i dont explain everything as good as i could.
i uploaded an output so you can see my problem: https://files.fm/u/vss3dd6v and if you select only the significant, they are very few..
thanks in advance..