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confusing cuffdifff results

Hi,

I got fully confused and frustrated by watching the result

I had 6 samples paired-end reads by tophat2

I used ensemble gtf and pre-index bowtie2

Left reads:
     Input   : 19667353
     Mapped  : 18672982 (94.9% of input)
      of these:  1127159 ( 6.0%) have multiple alignments (939 have >20)
Right reads:
     Input   : 19667353
     Mapped  : 17146234 (87.2% of input)
      of these:  1019710 ( 5.9%) have multiple alignments (848 have >20)
91.1% overall read mapping rate.

Aligned pairs: 16994149
  of these:  1010648 ( 5.9%) have multiple alignments
         25673 ( 0.2%) are discordant alignments
**86.3% concordant pair alignment rate.**

then I used cuffdiff to get differential expressed genes

This is my experiment details

In my experiment, before high temperature treatment (48 c), Arabidopsis cotyledons were pre-heated for 2, 8 and 24 hours. In the other hand I have cotyledons keeping in room temperature for 2, 8 and 24 hours, I mean without pre-heating these cotyledons directly face the high temperature. I don have any replication

Something like below

  • pre-heated-2h
  • pre-heated-8h
  • pre-heated-24h
  • unpre-heated-2h
  • unpre-heated-8h
  • unpre-heated-24h

I tried 3 cuffdiff syntaxs,but in ALL, in the gene_exp.diff file the significant column was totally NO, not only this file, the significant column wherever is NO , even when I labeled 6 samples, in the output I have only to columns for samples

cuffdiff -o diff_out -b genome.fa -p 8 \
    -L Pre-2h,unPre-2h,Pre-8h,unPre-8h,Pre-24h,unPre-24h \
    -u merged_asm/merged.gtf \
    -T pre-heated-2h.bam \
    unpre-heated-2h.bam pre-heated-8h.bam \
    unpre-heated-8h.bam pre-heated-24h.bam \
    unpre-heated-24h.bam #syntax1

cuffdiff -o diff_out -b genome.fa -p 8 \
    -L Pre-2h,unPre-2h,Pre-8h,unPre-8h,Pre-24h,unPre-24h \
    -u merged_asm/merged.gtf \
    pre-heated-2h.bam unpre-heated-2h.bam \
    pre-heated-8h.bam unpre-heated-8h.bam \
    pre-heated-24h.bam unpre-heated-24h.bam #syntax2

cuffdiff -o diff_out -b genome.fa -p 8 \
    -L pre,unpre \
    -u merged_asm/merged.gtf \
    pre-heated-2h.bam, pre-heated-8h.bam, \
    pre-heated-24h.bam unpre-heated-2h.bam \
    unpre-heated-8h.bam, unpre-heated-24h.bam #syntax3

How possible that there is no any differential expressed genes in my experiment??? :(

Thank you

EDIT (@RamRS): Code folder for better display

myposts rna-seq next-gen
• 1,470 views • link
updated by Ram Ram • written by zizigolu zizigolu

Hello Fereshteh!

Questions similar to yours can already be found at:

We have closed your question to allow us to keep similar content in the same thread.

If you disagree with this please tell us why in a reply below. We'll be happy to talk about it.

Cheers!

PS: Why open another question with the same content?

• 0 views • link
written by Ram Ram

It's very funny to try different commands to check differentially expressed genes. Let's say the command is wrong but you get differentially expressed genes (some junk), what do you do?

• 0 views • link
updated by Ram Ram • written by GouthamAtla GouthamAtla

You mean I did wrong commands? If so please tell me the right one

I should find some sense result to give to my supervisor. You can't imagine how much yesterday I cried, yesterday was one of the worst days in my life. She told she won't give me the datasets because I could not infer any results then without the data sets how I could defend my dissertation?

I know this is not sense to express such topic in this thread but you may know why I am asking silly questions

• 0 views • link
updated by Ram Ram • written by zizigolu zizigolu

What Goutham means is that you should judge your results by your approach, not the other way around. Data science will yield results if you keep tweaking your question, but along the way, your question itself will lose meaning. You'll get a flawed answer with no biological significance.

Your approach must be to ensure your command aligns with what you know of the data. And remember, no result is not research failing - it's research yielding a different yet important result - that there is no significant answer to your question. That does not make the question invalid, it just means that other questions need to be explored.

• 0 views • link
written by Ram Ram

Thank you Ram,

Actually when I saved the gene_exp.diff file as txt and inspected for any significance, I found more than 700 yes. now another step started. Anyway many thanks

• 0 views • link
updated by Ram Ram • written by zizigolu zizigolu

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