edgeR time series DMSO correction
1
0
Entering edit mode
aec ▴ 70
@aec-9409
Last seen 12 months ago

Dear all,

I have a time series RNA-seq experiment with a control at time 0 and would like to test the inhibitors effect over time (4h, 24h) but also want to correct for DMSO effect for each time (4h, 24h).

This is what I have:

sample

replica

cell

time

condition

cell_time_condition

1

R1

cell_line1

0h

BaseLine

cell_line1.0h.BaseLine

2

R1

cell_line1

4h

DMSO

cell_line1.4h.DMSO

3

R1

cell_line1

4h

INHIBITOR1

cell_line1.4h.INHIBITOR1

4

R1

cell_line1

4h

INHIBITOR2

cell_line1.4h.INHIBITOR2

5

R1

cell_line1

4h

COMBO

cell_line1.4h.COMBO

6

R1

cell_line1

24h

DMSO

cell_line1.24h.DMSO

7

R1

cell_line1

24h

INHIBITOR1

cell_line1.24h.INHIBITOR1

8

R1

cell_line1

24h

INHIBITOR2

cell_line1.24h.INHIBITOR2

9

R1

cell_line1

24h

COMBO

cell_line1.24h.COMBO

10

R2

cell_line1

0h

BaseLine

cell_line1.0h.BaseLine

11

R2

cell_line1

4h

DMSO

cell_line1.4h.DMSO

12

R2

cell_line1

4h

INHIBITOR1

cell_line1.4h.INHIBITOR1

13

R2

cell_line1

4h

INHIBITOR2

cell_line1.4h.INHIBITOR2

14

R2

cell_line1

4h

COMBO

cell_line1.4h.COMBO

15

R2

cell_line1

24h

DMSO

cell_line1.24h.DMSO

16

R2

cell_line1

24h

INHIBITOR1

cell_line1.24h.INHIBITOR1

17

R2

cell_line1

24h

INHIBITOR2

cell_line1.24h.INHIBITOR2

18

R2

cell_line1

24h

COMBO

cell_line1.24h.COMBO

19

R3

cell_line1

0h

BaseLine

cell_line1.0h.BaseLine

20

R3

cell_line1

4h

DMSO

cell_line1.4h.DMSO

21

R3

cell_line1

4h

INHIBITOR1

cell_line1.4h.INHIBITOR1

22

R3

cell_line1

4h

INHIBITOR2

cell_line1.4h.INHIBITOR2

23

R3

cell_line1

4h

COMBO

cell_line1.4h.COMBO

24

R3

cell_line1

24h

DMSO

cell_line1.24h.DMSO

25

R3

cell_line1

24h

INHIBITOR1

cell_line1.24h.INHIBITOR1

26

R3

cell_line1

24h

INHIBITOR2

cell_line1.24h.INHIBITOR2

27

R3

cell_line1

24h

COMBO

cell_line1.24h.COMBO

The comparisons I am interested would be:

0h vs 4h inhibitor1

0h vs 4h inhibitor2

0h vs 4h COMBO

...

but correcting for the DMSO effect.

Here a piece of the code:

design <- model.matrix(~0+group, data=y$samples)

my.contrasts <- makeContrasts(

cell_line1_0h_BaseLine_vs_cell_line1_4h_inhibitor1=(cell_line1.4h.inhibitor1-cell_line1.0h.BaseLine)-(cell_line1.4h.DMSO-cell_line1.0h.BaseLine),
cell_line1_0h_BaseLine_vs_cell_line1_4h_inhibitor2=(cell_line1.4h.inhibitor2-cell_line1.0h.BaseLine)-(cell_line1.4h.DMSO-cell_line1.0h.BaseLine),
cell_line1_0h_BaseLine_vs_cell_line1_4h_COMBO=(cell_line1.4h.COMBO-cell_line1.0h.BaseLine)-(cell_line1.4h.DMSO-cell_line1.0h.BaseLine),

levels=design)

 

where group="cell_time_condition".

Is the correction by DMSO correctly done in my contrasts?

edger makecontrasts timecourse • 691 views
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0
Entering edit mode
@ryan-c-thompson-5618
Last seen 9 months ago
Scripps Research, La Jolla, CA

This looks correct to me, but over-complicated. Notice that in each of your contrasts, the 2 cell_line1.0h.BaseLine terms cancel each other out.

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0
Entering edit mode

so how can I easily approach that?

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1
Entering edit mode

I think you need to be more precise in defining what you want to test. You've written all your contrasts in the form (4h.treat - 0h) - (4h.control - 0h), which simplifies to 4h.treat - 4h.control. This is the difference between the 4h time point with and without the treatment. This is the only contrast that makes sense to me. Since the initial condition is the same for both control and treatment, the effect of the treatment at a given timepoint is represented by the differences between control and treatment at that time point. It might be useful to also test the contrast representing what happens to the control over 4 hours, i.e. 4h.control - 0h. You can then compare the direction of change relative to the 4h treatment effect to this to see if the treatment is reinforcing or opposing the changes that would normally occur in those 4 hours.

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0
Entering edit mode

So apart from testing independently these two contrats:

4h.treat - 4h.control

4h.control - 0h

There is no other way to test the treatment effect (respect to time 0h) corrected by any effect of the DMSO (respect to time 0h)  at the same time? 

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0
Entering edit mode

I think you're misunderstanding Ryan. It's not that there's no way to test for differences in the "corrected" effects. It's that there's no point in testing them, because mathematically, the time 0h baseline will cancel out; so you might as well directly test for differences between the 4h treatment and DMSO groups, i.e., 4h.treat - 4h.control.

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0
Entering edit mode

ok, thanks.

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