Hello all,

I am new to RNA-seq, and would like your input to see if my results are real or not. I am trying to see if there are any differentially expressed genes between knockout and wild-type strain with 3 animals for each genotype. In my gene_exp.diff output from cuffdiff the most significant p-value is 5.00E-05 and I have 58 genes with this value, and all of them have the exact same q-value of 0.0150853. I am a bit skeptical that all of these genes can have the exact same p and q values....

Could it be that they actually have a lower significant value, but cuffdiff cuts them all off at a p-value of 5.00E-05 and q-value of 0.0150853?

I have included a sample of my results below & I am using cufflinks 2.2.1

I would appreciate any insight into this phenomenon.

Cheers,  
Yuka

```
value_1      value_2      log2(fold_change)     test_stat     p_value      q_value       significant
89.2154      58.9422      -0.597993             -2.83732      5.00E-05     0.0150853     yes
1.11605      0.637795     -0.807238             -2.87108      5.00E-05     0.0150853     yes
3.28464      2.19358      -0.582446             -2.43504      5.00E-05     0.0150853     yes
133.831      3.77205      -5.14892              -10.0845      5.00E-05     0.0150853     yes
439.124      707.344      0.687783              3.06388       5.00E-05     0.0150853     yes
46.5894      20.684       -1.17148              -4.54961      5.00E-05     0.0150853     yes
4.29346      0.487185     -3.1396               -5.51822      5.00E-05     0.0150853     yes
3.98649      2.53882      -0.650961             -2.34521      5.00E-05     0.0150853     yes
2.43337      7.40836      1.6062                3.70451       5.00E-05     0.0150853     yes
3.09364      1.61501      -0.937765             -2.6357       5.00E-05     0.0150853     yes
22.7107      34.5983      0.607328              2.75673       5.00E-05     0.0150853     yes
1.62867      0.384298     -2.0834               -3.16599      5.00E-05     0.0150853     yes
85.5877      40.7053      -1.07218              -3.79772      5.00E-05     0.0150853     yes
7.59285      4.52762      -0.74589              -3.00281      5.00E-05     0.0150853     yes
0.655639     1.15038      0.811131              3.2065        5.00E-05     0.0150853     yes
16.8931      9.04287      -0.901585             -3.10804      5.00E-05     0.0150853     yes
2.13982      1.15785      -0.886043             -2.26105      5.00E-05     0.0150853     yes
51.0117      70.4272      0.465303              2.23548       5.00E-05     0.0150853     yes
16.4426      4.60742      -1.8354               -6.30806      5.00E-05     0.0150853     yes
1.70664      3.06055      0.842634              2.63646       5.00E-05     0.0150853     yes
25898.9      11368.1      -1.1879               -2.67136      5.00E-05     0.0150853     yes
31.0799      43.4487      0.483332              2.38779       5.00E-05     0.0150853     yes
1.34841      0.854922     -0.657398             -2.31314      5.00E-05     0.0150853     yes
8.65166      2.89657      -1.57863              -6.03995      5.00E-05     0.0150853     yes
84.8254      128.85       0.603121              3.00014       5.00E-05     0.0150853     yes
7.3413       4.40212      -0.737838             -3.1716       5.00E-05     0.0150853     yes
35.9883      52.4989      0.544761              2.75034       5.00E-05     0.0150853     yes
5.09062      3.22742      -0.657457             -2.46845      5.00E-05     0.0150853     yes
3.54614      2.26065      -0.649511             -2.44616      5.00E-05     0.0150853     yes
5.88631      2.35884      -1.31929              -2.90402      5.00E-05     0.0150853     yes
1.63451      0.707828     -1.20739              -3.0934       5.00E-05     0.0150853     yes
7.81814      12.558       0.68371               2.81042       5.00E-05     0.0150853     yes
73.4685      110.3        0.586236              2.84624       5.00E-05     0.0150853     yes
3.8276       2.46574      -0.634417             -2.42448      5.00E-05     0.0150853     yes
22.13        14.2849      -0.631518             -2.55388      5.00E-05     0.0150853     yes
10.0575      5.06492      -0.989659             -3.61907      5.00E-05     0.0150853     yes
4.56879      2.70737      -0.754918             -2.5256       5.00E-05     0.0150853     yes
5.17134      3.10769      -0.734697             -2.88216      5.00E-05     0.0150853     yes
10.8373      6.79769      -0.672893             -2.30828      5.00E-05     0.0150853     yes
1.13635      0.563153     -1.0128               -2.01683      5.00E-05     0.0150853     yes
45.5072      76.8825      0.756562              3.63131       5.00E-05     0.0150853     yes
1.01017      0.112521     -3.16633              -4.40682      5.00E-05     0.0150853     yes
1.3256       0            #NAME?                #NAME?        5.00E-05     0.0150853     yes
3.47888      0            #NAME?                #NAME?        5.00E-05     0.0150853     yes
76.2555      33.5566      -1.18424              -4.46786      5.00E-05     0.0150853     yes
11.6164      19.7901      0.768611              3.5777        5.00E-05     0.0150853     yes
4.70657      3.21184      -0.551276             -2.49339      5.00E-05     0.0150853     yes
4.45358      1.18755      -1.90698              -5.88499      5.00E-05     0.0150853     yes
179.376      122.177      -0.554005             -2.59482      5.00E-05     0.0150853     yes
1.88054      1.23137      -0.610883             -2.60224      5.00E-05     0.0150853     yes
51.2297      34.7039      -0.561882             -2.35145      5.00E-05     0.0150853     yes
1.55802      5.58375      1.84152               4.20437       5.00E-05     0.0150853     yes
1.15313      0            #NAME?                #NAME?        5.00E-05     0.0150853     yes
6.63245      12.3112      0.89236               3.63541       5.00E-05     0.0150853     yes
4.33681      7.43317      0.777344              2.41337       5.00E-05     0.0150853     yes
57.6986      11.8767      -2.2804               -4.98563      5.00E-05     0.0150853     yes
6.55491      2.75186      -1.25217              -2.86793      5.00E-05     0.0150853     yes
25.3647      16.1568      -0.650683             -2.48292      5.00E-05     0.0150853     yes
```
<p>If I remember correctly, cuffdiff uses (in recent versions like yours) a kind of permutation test (<strong>edit</strong>: sampling procedure) to assess the significance of DE. This is why it has got &quot;bins&quot; of qvalues and the lowest one is still not so low. I&#39;m anyway not sure of this.</p>

<p>Whatever the cause of this is, it is a common situation, so it doesn&#39;t mean you did something wrong.</p>

<p><strong>edit:</strong> just found a very partial comment of the authours on this topic: see the last point of the description of 2.1.0 <a href="http://cole-trapnell-lab.github.io/cufflinks/releases/v2.1.0/">release</a></p>
