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

-- 
Rong-Lin Wang
National Exposure Lab
Cincinnati, Ohio, USA

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