Is there a limit to the p-value & q-value for cuffdiff gene_exp.diff?

Question

[Yuka Takemon]

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

[Devon Ryan]

Which version is this? I know there was an issue with this until one of the most recent versions.

[Yuka Takemon]

Oops I should have included that above: I am using cufflinks/2.2.1

[Fabio Marroni]

With cufflinks 2.2.0 I have the same problem. I didn't have it with older versions (I can't find the versions right now, sorry!). I wonder if the problem emerged when they started using the method of Simon and Anders for estimating dispersion across replicates.

[Yuka Takemon]

I am using 2.2.1

Answer 1

[Martombo]

If I remember correctly, cuffdiff uses (in recent versions like yours) a kind of permutation test (edit: sampling procedure) to assess the significance of DE. This is why it has got "bins" of qvalues and the lowest one is still not so low. I'm anyway not sure of this.

Whatever the cause of this is, it is a common situation, so it doesn't mean you did something wrong.

edit: just found a very partial comment of the authours on this topic: see the last point of the description of 2.1.0 release

[Martombo]

In any case, I would advise you against using cufflinks for almost any purpose. It has been shown to have a very low performance. I'd rather use kallisto for a transcript level analysis.

[Yuka Takemon]

Thanks for finding the 2.1.0 release... what a bummer. I guess I will have to learn Kallisto & Sleuth now.

[super]

I found when I did cuffdiff, lots of DE genes have same Q-value as well.Could this pipeline for Gene level, I think Kallisto & Sleuth is only suitable for DE transcripts, am I right?