[BioC] package LIMMA design matrix for my experiment

[Ryan C. Thompson]

If you're not sure how to proceed with the full experiment, I recommend 
that you start with a reduced design and figure out how to model it, and 
then progressively add complexity little by little. For your data, I 
would start by ignoring site, year, and season, and just figure out how 
to do a simple comparison of treatment vs control while dealing with the 
dye effects. Once you have a reasonable design for that, you can start 
figuring out how to add in the other experimental variables.

Unfortunately I can't give you any more specific advice because I have 
no experience with modelling two-color arrays with limma.

-Ryan Thompson

On 02/14/2013 07:39 PM, RLW wrote:
> Dear Limma users,
>
> Your suggestions,comments, thoughts appreciated on this posting.
>
> Here is an experimental design with 2 sites (D or Z), 2 years (08 or 10), 2
> seasons (S or F), and treated/control.  Samples receiving various
> combinations of these factors are placed in either Cy5 or Cy3.  There are 4
> types of controls, one for each season-year: S_08_time0, F_08_time0,
> S_10_time0, and F_10_time0.  Some of control samples are technical
> duplicates and marked as *_dup.  See target file below.
>
> One school of thought considers this as a factorial design while another
> regards it as a nested anova (i.e., all other factors nested under one of
> the two sites).  My belief is I can do neither because of a lack of common
> reference control across ALL 8 treatment combinations.  While the four
> types of controls are basically the same strain/species of untreated
> organisms, they came however from different batch of lab organisms in
> different season/year so are likely different to some extent.
>
> Here are my thoughts:
> 1. analyze each treatment group against its own control separately, namely:
>   D_F_08 and Z_F_08 against F_08_time0;
>   D_S_08 and Z_S_08 against S_08_time0;
>   D_F_10 and Z_F_10 against F_10_time0;
>   D_S_10 and Z_S_10 against S_10_time0
>
> This way, the impact of season, year, and site on treated/control can only
> be inferred indirectly.
>
> 2. technical duplicates in the controls should be excluded, while all
> treated ones are unique biological samples and should be kept.  This would
> make sample size unbalanced within each of eight treatment groups.  Does
> this mean I have to analyze my data in single channels in order to exclude
> the technical duplicate controls from either Cy5 or Cy3?  The limma user
> guide (page 88) gave an example involving a composite design (reference and
> direct comparison) where all treated samples were compared to the pooled
> control only.  The authors used log ratio in that case.
>
> 3. How should we account for Cy5 vs Cy3 correlation if we are going to
> compare a few treated samples from either Cy5 or Cy3 against control
> samples also from either channel?  These samples are not necessarily from
> the same arrays.
>
> Thanks for your feedback!
>
>
> Name    Cy3    Cy5    Cy3SampleName    Cy5SampleName
> 330_1_2    D-F-08    F-08_Time0_dup    9_22_08_D_L7    9_22_08_T0_L6
> 464_1_2    D-F-08    F-08_Time0    9_22_08_D_L6    9_22_08_T0_L3
> 331_2_2    F-08_Time0    D-F-08    9_22_08_T0_L2    9_22_08_D_L2
> 422_2_2    F-08_Time0_dup    D-F-08    9_22_08_T0_L3    9_22_08_D_L3
> 423_2_4    F-08_Time0    D-F-08    9_22_08_T0_L6    9_22_08_D_L4
> 328_2_4    D-F-10    F-10_Time0_dup    9_14_10_D_L1    9_14_10_T0_L1
> 329_1_4    D-F-10    F-10_Time0    9_14_10_D_L2    9_14_10_T0_L2
> 330_1_1    D-F-10    F-10_Time0    9_14_10_D_L3    9_14_10_T0_L3
> 422_1_4    F-10_Time0_dup    D-F-10    9_14_10_T0_L2    9_14_10_D_L5
> 423_1_3    F-10_Time0_dup    D-F-10    9_14_10_T0_L3    9_14_10_D_L6
> 464_2_1    F-10_Time0    D-F-10    9_14_10_T0_L1    9_14_10_D_L4
> 328_1_1    D-S-08    S-08_Time0_dup    6_3_08_D_L2    6_3_08_T0_L1
> 464_1_3    D-S-08    S-08_Time0    6_3_08_D_L4    6_3_08_T0_L6
> 464_1_4    D-S-08    S-08_Time0    6_3_08_D_L5    6_3_08_T0_L8
> 331_1_4    S-08_Time0    D-S-08    6_3_08_T0_L1    6_3_08_D_L6
> 423_2_1    S-08_Time0_dup    D-S-08    6_3_08_T0_L8    6_3_08_D_L8
> 329_2_2    D-S-10    S-10_Time0_dup    5_25_10_D_L2    5_25_10_T0_L3
> 330_1_3    D-S-10    S-10_Time0    5_25_10_D_L3    5_25_10_T0_L4
> 464_1_1    D-S-10    S-10_Time0    5_25_10_D_L1    5_25_10_T0_L1
> 331_2_4    S-10_Time0_dup    D-S-10    5_25_10_T0_L1    5_25_10_D_L4
> 422_2_3    S-10_Time0    D-S-10    5_25_10_T0_L3    5_25_10_D_L5
> 423_2_3    S-10_Time0_dup    D-S-10    5_25_10_T0_L4    5_25_10_D_L6
> 331_1_3    F-08_Time0_dup    Z-F-08    9_15_08_T0_L1    9_22_08_Z_L4
> 422_2_1    F-08_Time0    Z-F-08    9_15_08_T0_L2    9_22_08_Z_L5
> 423_1_2    F-08_Time0    Z-F-08    9_15_08_T0_L3    9_22_08_Z_L7
> 328_1_3    Z-F-08    F-08_Time0    9_22_08_Z_L1    9_15_08_T0_L1
> 329_1_2    Z-F-08    F-08_Time0_dup    9_22_08_Z_L2    9_15_08_T0_L2
> 330_2_1    Z-F-08    F-08_Time0_dup    9_22_08_Z_L3    9_15_08_T0_L3
> 331_1_2    F-10_Time0_dup    Z-F-10    9_7_10_T0_L4    9_14_10_Z_L4
> 422_2_4    F-10_Time0    Z-F-10    9_7_10_T0_L5    9_14_10_Z_L5
> 423_2_2    F-10_Time0_dup    Z-F-10    9_7_10_T0_L6    9_14_10_Z_L6
> 328_2_1    Z-F-10    F-10_Time0    9_14_10_Z_L1    9_7_10_T0_L4
> 329_2_3    Z-F-10    F-10_Time0_dup    9_14_10_Z_L2    9_7_10_T0_L5
> 330_2_4    Z-F-10    F-10_Time0    9_14_10_Z_L3    9_7_10_T0_L6
> 331_2_1    S-08_Time0_dup    Z-S-08    5_27_08_T0_L6    6_3_08_Z_L6
> 422_1_1    S-08_Time0    Z-S-08    5_27_08_T0_L7    6_3_08_Z_L7
> 328_2_3    Z-S-08    S-08_Time0    6_3_08_Z_L1    5_27_08_T0_L6
> 329_1_1    Z-S-08    S-08_Time0_dup    6_3_08_Z_L2    5_27_08_T0_L7
> 330_1_4    Z-S-08    S-08_Time0    6_3_08_Z_L3    5_27_08_T0_L8
> 423_1_1    S-10_Time0_dup    Z-S-10    5_18_10_T0_L6    5_25_10_Z_L7
> 464_2_2    S-10_Time0    Z-S-10    5_18_10_T0_L3    5_25_10_Z_L5
> 464_2_4    S-10_Time0    Z-S-10    5_18_10_T0_L4    5_25_10_Z_L6
> 328_1_2    Z-S-10    S-10_Time0_dup    5_25_10_Z_L2    5_18_10_T0_L3
> 329_2_4    Z-S-10    S-10_Time0_dup    5_25_10_Z_L3    5_18_10_T0_L4
> 330_2_3    Z-S-10    S-10_Time0    5_25_10_Z_L4    5_18_10_T0_L6
>