Hi,

I have a couple quick questions regarding experimental setup and analysis. I am trying to figure out how to best design my experiment to get the information I need while also minimizing cost. I am using an inducible transgene to identify the targets of my transcription factor with mRNA-seq. I am setting up my experiment with a two- way ANOVA design:

Inducible Line (not treated) ; Inducible line (treated with inducing chemical)

Wild-type control (not treated); Wild-type control (treated with inducing chemical)

At a minimum, I plan to submit two biological replicates for sequencing for both lines/conditions. I am also hoping to using additional time points post induction (again, will depend on cost).

Question 1) I know more replicates are better, but there is the cost limitation. How much will my statistical power increase by including a 3rd replicate? Is it likely that just two replicates would be useful or is this something that needs to be determined empirically? I have read the other threads on the website about the Fisher's Exact Test (two replicates) vs. T-test (need 3 replicates), but don't know if the value of the 3rd replicate similarly holds true for the two-way ANOVA.

Question 2) When I have my data, I will need software to accommodate the two-way ANOVA analysis. Do most NGS analysis software packages offer two-way ANOVA analysis? Does anyone have any recommendations? I am working with Arabidopsis.

If anyone has any insight into either of these questions, I would be grateful for your input. Thanks!

eggplant72

I have a couple quick questions regarding experimental setup and analysis. I am trying to figure out how to best design my experiment to get the information I need while also minimizing cost. I am using an inducible transgene to identify the targets of my transcription factor with mRNA-seq. I am setting up my experiment with a two- way ANOVA design:

Inducible Line (not treated) ; Inducible line (treated with inducing chemical)

Wild-type control (not treated); Wild-type control (treated with inducing chemical)

At a minimum, I plan to submit two biological replicates for sequencing for both lines/conditions. I am also hoping to using additional time points post induction (again, will depend on cost).

Question 1) I know more replicates are better, but there is the cost limitation. How much will my statistical power increase by including a 3rd replicate? Is it likely that just two replicates would be useful or is this something that needs to be determined empirically? I have read the other threads on the website about the Fisher's Exact Test (two replicates) vs. T-test (need 3 replicates), but don't know if the value of the 3rd replicate similarly holds true for the two-way ANOVA.

Question 2) When I have my data, I will need software to accommodate the two-way ANOVA analysis. Do most NGS analysis software packages offer two-way ANOVA analysis? Does anyone have any recommendations? I am working with Arabidopsis.

If anyone has any insight into either of these questions, I would be grateful for your input. Thanks!

eggplant72

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