<p>Depending upon the type of treatment used the set of DEGs will change.If the treatments have similar kind of effect you will get a small list (less variable genes will have higher p-val) using a cutoff of p&lt;0.05. So, it's better to start with control vs treated comparisons then T1 vs T2.</p>

<p>Compare the lists form CTRL vs T1 and CTRL vs T2 you will get the genes that are expressed in both conditions as well as unique to individual treatments.</p>

CC BY-SA 3.0
2017-10-31T07:12:25.700Z
If a gene has a p-value of 0.049 in CTRL vs. T1 and 0.05 in CTRL vs. T2 you would be categorizing it as uniquely DE in one condition, when there's no actual difference between the conditions.
Devon Ryan
CC BY-SA 3.0
2017-10-31T13:45:56.200Z
Yes, it's a tricky situation.
arup
CC BY-SA 3.0
2017-10-31T16:49:44.040Z
I agree it is a trick situation in my case it is modes of exercise so they are extremely similar responses. The T1 vs T2 comparison yields almost nothing where the vs baseline comparisons are quite different.
Nitro
CC BY-SA 3.0
2017-10-31T05:19:17.837Z
2017-10-31T05:19:17.837Z
arup
<p>If you're interested in looking at the differences between two treatments then you'll end up wanting to do both a direct contrast as well as the individual comparisons to baseline.</p>

<p>The direct contrast will give you the genes actually differentially expressed between the two conditions. In practice, you may want to filter this a bit so you only have genes differentially expressed vs. baseline in at least one condition (e.g., to get rid of genes only slightly higher due to T1 and slightly lower in T2, but not different enough in either case to be DE). Use a pretty lax p-value threshold (e.g., 0.1 or 0.2) for this.</p>

CC BY-SA 3.0
2017-10-30T20:05:26.650Z
Thanks. What is your opinion on genes that may be significant in just 1 of the comparisons to baseline but not significant in the direct T1 vs T2 contrast?
Nitro
CC BY-SA 3.0
2017-10-30T20:22:56.303Z
It there's no difference in the T1 vs. T2 contrast then there's no difference. It doesn't matter if you happen to get a significant difference in one of the conditions vs. baseline. You only want to use the comparisons to baseline for filtering your T1 vs. T2 results (assuming you have enough DE genes to bother doing so).
Devon Ryan
CC BY-SA 3.0
2017-10-30T21:21:12.480Z
One more question. So if I wanted to do a pathway analysis and compare the two treatments that way. When I generate my gene lists how should I treat genes that are DE in only one treatment vs baseline but not DE in the direct T1 vs T2 contrast?
Nitro
CC BY-SA 3.0
2017-10-30T21:24:37.050Z
Treat them as not DE.
Devon Ryan
CC BY-SA 3.0
2017-10-30T21:31:39.077Z
So any genes that have similar responses from baseline in both treatments get discarded? Wouldn't you be missing a lot of context if a gene has to be DE vs baseline in both treatments and also DE directly with each other to make either final list?
Nitro
CC BY-SA 3.0
2017-10-30T21:37:14.993Z
Yes, any genes with similar responses should get discarded, since they're not different. A gene doesn't have to be DE in either treatment vs. baseline to be DE between them. That's also why I suggested using a very lax p-value threshold if you want to do any filtering of the T2 vs. T1 results.
Devon Ryan
CC BY-SA 3.0
2017-10-30T22:02:53.920Z
Sorry, I meant doing a pathway analysis of each treatment vs baseline separately to find the raw response of each treatment. Since they are different from baseline I would still include them in only their respective pathway analysis?
Nitro
CC BY-SA 3.0
2017-10-30T22:06:39.160Z
Yes, certainly include them in such cases.
Devon Ryan
CC BY-SA 3.0
2017-10-30T22:09:15.387Z
Cool, thanks a lot for your help. I really appreciate it.
Nitro
CC BY-SA 3.0
2017-10-30T19:50:47.230Z
2017-10-30T19:50:47.230Z
Devon Ryan
<p>I have a expression data from a small cohort of samples taken at baseline and after 2 independent treatments. I can do differential expression contrasting T1 and T2 or I can contrast T1 vs baseline and T2 vs baseline and look at the differences. What is the difference between these two analyses? Are they both valid? If so what inferences can be drawn from each?</p>

<differential-expression>
Differential Expression With 2 Treatments
# Differential Expression With 2 Treatments

(Asked by: username_0 on 2017-10-30)

I have a expression data from a small cohort of samples taken at baseline and after 2 independent treatments. I can do differential expression contrasting T1 and T2 or I can contrast T1 vs baseline and T2 vs baseline and look at the differences. What is the difference between these two analyses? Are they both valid? If so what inferences can be drawn from each?

**Tags:** differential-expression

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## 2 answers

### Answer by username_2 on 2017-10-30. Score: 4 | Accepted answer

If you're interested in looking at the differences between two treatments then you'll end up wanting to do both a direct contrast as well as the individual comparisons to baseline.

The direct contrast will give you the genes actually differentially expressed between the two conditions. In practice, you may want to filter this a bit so you only have genes differentially expressed vs. baseline in at least one condition (e.g., to get rid of genes only slightly higher due to T1 and slightly lower in T2, but not different enough in either case to be DE). Use a pretty lax p-value threshold (e.g., 0.1 or 0.2) for this.

> **Comments:**
> **username_0**: Thanks. What is your opinion on genes that may be significant in just 1 of the comparisons to baseline but not significant in the direct T1 vs T2 contrast?  
> **username_2**: It there's no difference in the T1 vs. T2 contrast then there's no difference. It doesn't matter if you happen to get a significant difference in one of the conditions vs. baseline. You only want to use the comparisons to baseline for filtering your T1 vs. T2 results (assuming you have enough DE genes to bother doing so).  
> **username_0**: One more question. So if I wanted to do a pathway analysis and compare the two treatments that way. When I generate my gene lists how should I treat genes that are DE in only one treatment vs baseline but not DE in the direct T1 vs T2 contrast?  
> **username_2**: Treat them as not DE.  
> **username_0**: So any genes that have similar responses from baseline in both treatments get discarded? Wouldn't you be missing a lot of context if a gene has to be DE vs baseline in both treatments and also DE directly with each other to make either final list?  
> **username_2**: Yes, any genes with similar responses should get discarded, since they're not different. A gene doesn't have to be DE in either treatment vs. baseline to be DE between them. That's also why I suggested using a very lax p-value threshold if you want to do any filtering of the T2 vs. T1 results.  
> **username_0**: Sorry, I meant doing a pathway analysis of each treatment vs baseline separately to find the raw response of each treatment. Since they are different from baseline I would still include them in only their respective pathway analysis?  
> **username_2**: Yes, certainly include them in such cases.  
> **username_0**: Cool, thanks a lot for your help. I really appreciate it.  

---

### Answer by username_1 on 2017-10-31. Score: 3

Depending upon the type of treatment used the set of DEGs will change.If the treatments have similar kind of effect you will get a small list (less variable genes will have higher p-val) using a cutoff of p<0.05. So, it's better to start with control vs treated comparisons then T1 vs T2.

Compare the lists form CTRL vs T1 and CTRL vs T2 you will get the genes that are expressed in both conditions as well as unique to individual treatments.

> **Comments:**
> **username_2**: If a gene has a p-value of 0.049 in CTRL vs. T1 and 0.05 in CTRL vs. T2 you would be categorizing it as uniquely DE in one condition, when there's no actual difference between the conditions.  
> **username_1**: Yes, it's a tricky situation.  
> **username_0**: I agree it is a trick situation in my case it is modes of exercise so they are extremely similar responses. The T1 vs T2 comparison yields almost nothing where the vs baseline comparisons are quite different.  

---

