Hi

I am seeking some clarification on a design matrix for edgeR.

My experimental design is as follows:

2 groups (knock in & knock out, n=7 per group), within each group I have paired samples from the same animal (vector, empty vector).

The contrasts I would like to make are:

• knock in v knock in empty vector
• knock out v knock out empty vector
• empty vector v empty vector

My current design matrix looks like this:

des <- model.matrix(~ manip + manip:mouse + manip:vector, data=targets)

Where the 'targets' file looks like: <style type="text/css"> .tg {border-collapse:collapse;border-spacing:0;} .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} .tg .tg-yw4l{vertical-align:top} </style>

where manip is knock in or knock out, rat is the specific animal and vector is the active or empty vector.

Using this design matrix I can extract the manip regulated genes with: ki_cont <- makeContrasts('manipknock.in.vectorvector', levels=design) ko_cont <- makeContrasts('manipknock.out.vectorvector', levels=design)

These look good as far as gene lists go.

My questions are:

• Do the above contrasts account for the pair information I have?

• How do I go about extracting the information to compare empty vectors under both manipulations?

Thanks for any help or advice.

Best,

iain