Off topic:confusing cuffdifff results

Question

[zizigolu]

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