text: user3056: I have recently started working on a substance's effect on a cell line in different dosages. for this, there is a tool called bmdexpress2 that I am using. Its input is the normalized counts from RNASeq for each dosage as a big matrix. When it comes to the pathway analysis step, unlike hallmarks of GSEA, this tool uses some databases for defining pathways which involve pathways of even 2-3 genes.
  So what I want to discuss here is the strength of small pathways and filtering thresholds. How can we bioinformatically decide on the importance of a pathway made of 3 genes? Should we just filter them out as there are much more comprehensive pathways? Or do their GO Levels matter? Also there are many cases that out of those 3 genes, 1 of them is differentially expressed; is having 1 gene diff. expressed out of X genes in a pathway enough to say that the pathway is enriched?
  Here is an example GO term for a small pathway.
  And here is an example of the output I get from the tool. all genes platform column is the number of genes involved in that pathway, input genes column is how many of the genes in that pathway are significantly differentiated. I believe the rest is self explanatory. 
  
  Looking at the table, can we really say, ESCRT III Complex, for example is a good pathway to focus while having 1 gene out of 13 in a pathway but that change is significant?
  Thanks
  
  user581: The problem here is that any one GO term with a small number of members is unlikely to include any differentially expressed genes by chance, but if you test a large number of GO terms, some of them definitley will. The solution is to correct the p-values for multiple comparisons. If you do this, then none of those GO terms with a single DE member will be significant. The larger GO terms with multiple DE members in the chart you show probably won't survive correction either. Hopefully you have some that are more significant.
  
original_text: 3056: <p>I have recently started working on a substance's effect on a cell line in different dosages. for this, there is a tool called <a href="https://www.sciome.com/bmdexpress/" rel="nofollow noreferrer">bmdexpress2</a> that I am using. Its input is the normalized counts from RNASeq for each dosage as a big matrix. When it comes to the pathway analysis step, unlike hallmarks of GSEA, this tool uses some databases for defining pathways which involve pathways of even 2-3 genes.</p>
  
  <p>So what I want to discuss here is the strength of small pathways and filtering thresholds. How can we bioinformatically decide on the importance of a pathway made of 3 genes? Should we just filter them out as there are much more comprehensive pathways? Or do their GO Levels matter? Also there are many cases that out of those 3 genes, 1 of them is differentially expressed; is having 1 gene diff. expressed out of X genes in a pathway enough to say that the pathway is enriched?</p>
  
  <p>Here is an <a href="https://www.ebi.ac.uk/QuickGO/term/GO:0001966" rel="nofollow noreferrer">example</a> GO term for a small pathway.</p>
  
  <p>And here is an example of the output I get from the tool. all genes platform column is the number of genes involved in that pathway, input genes column is how many of the genes in that pathway are significantly differentiated. I believe the rest is self explanatory. </p>
  
  <p><a href="https://i.stack.imgur.com/QFaPd.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/QFaPd.png" alt="enter image description here"></a></p>
  
  <p>Looking at the table, can we really say, ESCRT III Complex, for example is a good pathway to focus while having 1 gene out of 13 in a pathway but that change is significant?</p>
  
  <p>Thanks</p>
   581: <p>The problem here is that any one GO term with a small number of members is unlikely to include any differentially expressed genes by chance, but if you test a large number of GO terms, some of them definitley will. The solution is to correct the p-values for <a href="https://en.wikipedia.org/wiki/Multiple_comparisons_problem" rel="nofollow noreferrer">multiple comparisons</a>. If you do this, then none of those GO terms with a single DE member will be significant. The larger GO terms with multiple DE members in the chart you show probably won't survive correction either. Hopefully you have some that are more significant.</p>
  