Different results at GO:BP

ExpressAnalyst
1

Hi,
A few days ago I uploaded a list of gene_symbols (see attached file) and got 120 pathways with FDR<0.05.
When I’ m doing the same thing today I get 0 pathways with FDR<0.05.
gene_symbol_list.txt (52.0 KB)

Even when the Hits are higher than the Expected, What happened ?
Here is the new list:

Pathway Total Expected Hits P.Value FDR
Axonogenesis 578 284 321 0.000862 0.707
Axon guidance 394 193 220 0.00373 1
Regulation of neurogenesis 444 218 246 0.00384 1
Regulation of synapse structure and activity 56 27.5 36 0.0155 1
Negative regulation of DNA binding 37 18.2 25 0.0178 1
Microtubule_based movement 179 87.8 102 0.0198 1
Gliogenesis 176 86.4 100 0.023 1
Neurotransmitter secretion 108 53 63 0.033 1
Regulation of endocytosis 128 62.8 73 0.0425 1
Regulation of axonogenesis 104 51 60 0.0476 1
Glutamate receptor signaling pathway 52 25.5 32 0.0478 1

and this is the older list from few days ago:

Pathway Total Expected Hits P.Value FDR
Axonogenesis 578 211 321 2.51E-21 2.06E-18
Neuron differentiation 1190 434 584 9.41E-21 3.86E-18
Neuron projection development 816 299 423 4.23E-20 1.16E-17
Neuron development 945 346 478 7.18E-20 1.47E-17
Generation of neurons 1300 477 624 1.60E-18 2.24E-16
Neurogenesis 1390 509 660 1.64E-18 2.24E-16
Regulation of neurogenesis 444 162 246 2.02E-16 2.36E-14
Axon guidance 394 144 220 2.63E-15 2.70E-13
Transmission of nerve impulse 871 319 426 1.15E-14 1.05E-12
Cell morphogenesis involved in differentiation 827 303 402 2.18E-13 1.79E-11
Synaptic transmission 765 280 375 3.41E-13 2.54E-11
Nervous system development 2190 802 943 1.04E-11 7.10E-10
Central nervous system development 784 287 374 3.69E-11 2.33E-09
Brain development 559 204 268 1.37E-08 8.03E-07
Microtubule_based movement 179 65.5 102 1.90E-08 1.04E-06
Gliogenesis 176 64.4 100 3.19E-08 1.64E-06
Cell_cell signaling 1310 478 564 1.74E-07 8.41E-06
Response to drug 344 126 172 1.85E-07 8.44E-06
Positive regulation of cellular component organization 560 205 260 7.06E-07 3.05E-05
Cell development 1840 675 766 1.45E-06 5.96E-05
Regulation of endocytosis 128 46.8 73 1.84E-06 7.17E-05
Neurotransmitter secretion 108 39.5 63 3.28E-06 0.000122
Behavior 597 218 271 3.98E-06 0.000142
Response to steroid hormone stimulus 267 97.7 133 5.65E-06 0.000193
Response to hypoxia 245 89.6 123 7.74E-06 0.000254
Regulation of axonogenesis 104 38 60 9.04E-06 0.000275
Synapse organization 161 58.9 86 8.89E-06 0.000275
Generation of a signal involved in cell_cell signaling 393 144 185 1.01E-05 0.000293
Regulation of neurotransmitter levels 157 57.4 84 1.04E-05 0.000293
Cellular component disassembly 310 113 150 1.14E-05 0.000312
Positive regulation of cell differentiation 571 209 257 1.51E-05 4.00E-04
Cellular protein complex assembly 343 125 163 1.75E-05 0.000447
Positive regulation of developmental process 817 299 355 1.90E-05 0.000472
Regulation of cell morphogenesis 325 119 155 2.19E-05 0.00052
Regulation of synapse structure and activity 56 20.5 36 2.28E-05 0.00052
Sensory organ development 485 177 221 2.26E-05 0.00052
Cellular protein complex disassembly 160 58.5 84 2.64E-05 0.000585
Protein complex disassembly 167 61.1 87 2.80E-05 0.000604
Regulation of mitotic cell cycle 351 128 165 3.27E-05 0.000687
Regulation of cytoskeleton organization 306 112 146 3.65E-05 0.000731
Regulation of membrane potential 306 112 146 3.65E-05 0.000731
Learning or memory 166 60.7 86 4.05E-05 0.000791
Regulation of cell migration 456 167 207 5.38E-05 0.00103
Mitotic cell cycle checkpoint 149 54.5 78 5.73E-05 0.00107
Negative regulation of cellular component organization 370 135 171 7.46E-05 0.00136
Cytoskeleton_dependent intracellular transport 76 27.8 44 0.000122 0.00212
Locomotory behavior 147 53.8 76 0.000121 0.00212
Negative regulation of DNA binding 37 13.5 25 0.000125 0.00212
Positive regulation of epithelial cell proliferation 120 43.9 64 0.000127 0.00212
Macromolecular complex disassembly 189 69.1 94 0.000135 0.00218
Positive regulation of cell adhesion 127 46.5 67 0.000137 0.00218
Regulation of anatomical structure morphogenesis 702 257 303 0.000138 0.00218
Regulation of DNA binding 70 25.6 41 0.000145 0.00224
Exocytosis 309 113 144 0.000167 0.00253
Regulation of action potential 169 61.8 85 0.000171 0.00255
Protein targeting to membrane 158 57.8 80 0.000199 0.00291
Neurological system process 1480 540 603 0.000211 0.00304
Glutamate receptor signaling pathway 52 19 32 0.000217 0.00307
Aging 197 72.1 96 0.000295 0.0041
Regulation of cell adhesion 294 108 136 0.00037 0.00505
Protein complex assembly 861 315 362 0.000377 0.00507
Developmental maturation 177 64.8 87 0.000388 0.00514
Regulation of cellular component organization 1520 558 618 0.000396 0.00515
DNA damage response, signal transduction by p53 class mediator 117 42.8 61 0.000405 0.00519
Tube development 506 185 221 0.000496 0.00626
Negative regulation of cell proliferation 585 214 252 0.000566 0.00703
S phase of mitotic cell cycle 144 52.7 72 0.000643 0.00787
Microtubule_based process 516 189 224 0.000682 0.00822
Cellular component assembly 1790 654 716 0.000717 0.00852
Actin filament bundle assembly 83 30.4 45 0.000765 0.00896
Response to abiotic stimulus 876 320 365 0.000776 0.00896
Second_messenger_mediated signaling 166 60.7 81 8.00E-04 0.00912
Actin filament organization 250 91.5 116 0.000835 0.00925
Synapse assembly 81 29.6 44 0.000826 0.00925
Signal transduction in response to DNA damage 129 47.2 65 0.000888 0.0097
Cell projection assembly 229 83.8 107 0.000964 0.0103
Cellular membrane organization 471 172 205 0.000963 0.0103
Cell_cell junction organization 186 68 89 0.001 0.0105
S phase 153 56 75 0.00104 0.0108
Positive regulation of hydrolase activity 497 182 215 0.00107 0.011
Maintenance of protein location 116 42.4 59 0.00112 0.0113
Regulation of binding 189 69.1 90 0.00114 0.0114
Response to hormone stimulus 751 275 314 0.00137 0.0136
Negative regulation of protein metabolic process 540 198 231 0.00146 0.0143
Protein polymerization 167 61.1 80 0.00168 0.0162
Secretion by cell 831 304 344 0.0018 0.0172
Cytoskeleton organization 980 359 401 0.00207 0.0195
Mitotic spindle organization 40 14.6 24 0.00216 0.0201
Positive regulation of cell migration 263 96.2 119 0.00221 0.0204
Negative regulation of cellular protein metabolic process 463 169 199 0.00232 0.0211
Cyclic_nucleotide_mediated signaling 36 13.2 22 0.00236 0.0213
Cell division 507 185 216 0.00259 0.023
Regulation of neuron apoptotic process 150 54.9 72 0.00261 0.023
Cellular component disassembly involved in execution phase of apoptosis 78 28.5 41 0.00279 0.0241
Negative regulation of binding 78 28.5 41 0.00279 0.0241
Regulation of cellular component size 192 70.2 89 0.00326 0.0279
Skeletal system development 459 168 196 0.00353 0.0298
Regulation of angiogenesis 169 61.8 79 0.00409 0.0342
G1/S transition of mitotic cell cycle 209 76.5 95 0.00493 0.0409
Maintenance of location 192 70.2 88 0.00504 0.041
Negative regulation of cytoskeleton organization 80 29.3 41 0.00505 0.041
Maintenance of protein location in cell 101 36.9 50 0.00518 0.0414
Positive regulation of angiogenesis 94 34.4 47 0.0052 0.0414
Morphogenesis of an epithelium 440 161 187 0.00544 0.0429
Establishment of organelle localization 125 45.7 60 0.00565 0.0441
Extracellular structure organization 242 88.5 108 0.00576 0.0445
Regulation of growth 548 200 229 0.00592 0.0454
Macromolecular complex assembly 1120 410 450 0.00606 0.046
Secretion 947 346 383 0.00613 0.0461
Intrinsic apoptotic signaling pathway 135 49.4 64 0.0062 0.0462
Response to external stimulus 1510 552 597 0.00654 0.0483
Peripheral nervous system development 81 29.6 41 0.00666 0.0488

Thank’s
Hagit

2

Overrepresentation analysis p-value for each pathway depends on 3 numbers:

  • Size of the entire ‘background universe’ - should be the list of all genes measured in the experiment
  • Size of the list of differentially expressed genes
  • Size of the pathway

Previously, for uploaded lists, the background was taken to be all genes in the selected pathway database, and the list of DEGs was filtered to remove any genes not in the pathway database. This ‘boosts’ the significance of all pathway results, giving more significant results and we have recently removed this filtering step to make the results more consistent across pathway databases. For most datasets, it should not result in a major change, however in your case there were >7000 DEGs and many were not in the pathway database. In general, ORA does not usually return significant results when there are so many DEGs. I recommend increasing the p-value and/or fold-change thresholds until you have fewer than 2500 DEGs, to investigate which pathways are overrepresented in the most strongly perturbed genes.

A heads up - we are further updating the pathway analysis for list upload to give users more control over the definition of the three numbers listed above. You can expect to see these changes in the next week or so.

1 Like
3

Thanks a lot, I will check again in few days.