Dear colleagues and dear Mark:

We want to extract the CPM from a DGElist object after TMM 
normalization, common and tagwise dispersion evaluation.

Here is a sample of the code, it's just a standard edgeR DEG workflow:

samplesheet1 <- read.delim("sample_sheet.txt", stringsAsFactors = FALSE)
comparison <- c("Tumor_1","Tumor_2")
RG1 <- readDGE(samplesheet1,header=FALSE)
colnames(RG1) <- gsub(".mature_cnt.txt","",RG1$sample$files,perl=TRUE)
N<-length(colnames(RG1))/2
K<-10
keep <- rowSums(cpm(RG1) > K) >= N
difflist1 <- RG1[keep,]
difflist1$samples$lib.size <- colSums(difflist1$counts)
difflist1 <- calcNormFactors(difflist1)
difflist1 <- estimateCommonDisp(difflist1)
difflist1 <- estimateTagwiseDisp(difflist1)
currentDiff <- difflist1
de.com <- exactTest(currentDiff,pair=comparison)

... then there is the creation of an Excel output dataframes, including 
the CPM worksheet:

addDataFrame(*round(cpm(currentDiff)*[,currentDiff$samples$group==comparison[1] 
| currentDiff$samples$group==comparison[2]]), ucpm_sheet, startRow=2, 
startColumn=1)

Now, the CPM content leaves us quite puzzled because it is very variable 
according to the syntax we use
to extract it, and this particular syntax seems to give values which do 
not sum up to 1 million as expected.
This is the syntax coherent with the

Apparently the only CPM values which do sum up correctly to 1 million es 
expected are those extracted with the following
instruction

head(cpm(currentDiff$counts))

But what are then these CPM values, which are different from the ones 
extracted with the command above ?

head(cpm(currentDiff))


These CPM values are different also from the values obtained with this 
command


head(cpm(currentDiff$pseudo.counts))


Many thanks in advance for any clarification !

Alessandro, Moreno & Paolo

----


>  head(cpm(currentDiff))

LT1C     LT2C   LT3C   ST2C    ST4C   ST10C   ST12C ST5C    ST7C    
ST8C   ST11C   LT1P    LT2P

hsa-miR-140-5p 306.550 209.4272 233.03 273.53 581.762 263.281 140.452 
285.775 633.920 679.240 232.223 396.27 203.828

hsa-miR-133a-3p 28.945  14.5648  51.61  44.04   4.116   5.495  33.441 
55.981   4.393  18.394   2.609  29.45   2.474

hsa-miR-665 11.695  21.7297  36.35  16.71   4.785  18.865  46.817 3.762  
12.213  19.308  20.162   3.22  32.849

hsa-miR-1250-5p 2.047   0.1175  13.03  10.21   3.344  13.462   3.541 
6.396   2.357   4.166   3.202  28.35   4.123

hsa-miR-381-3p 19.223  24.1963  84.53  44.74  16.104  10.898  33.834 
182.992  43.926  37.092   1.186  25.37  10.308

hsa-miR-374b-3p 39.543   7.6348  10.46  27.62  31.077  18.224  25.966 
17.457  39.747  29.572   7.353  22.46  13.607

LT3P   ST2P   ST10P  ST12P   ST7P    ST8P    ST4P   ST5P ST11P     SB

hsa-miR-140-5p 401.843 309.98 408.091 231.87 469.49 664.349 213.087 
551.31 870.579 326.37

hsa-miR-133a-3p 2.974  16.17   5.822  14.44  43.60  48.011  27.482  
27.06 13.031  41.21

hsa-miR-665 15.179  46.63  18.831  15.42   4.69   3.359   7.923  11.81 
3.425   4.57

hsa-miR-1250-5p 10.904  12.05  18.831  19.21  27.76   1.976  21.870  
48.22 67.411  12.49

hsa-miR-381-3p 11.957  44.73  10.007  16.40  21.45  23.907  80.795  
21.46 32.661  75.00

hsa-miR-374b-3p 30.048  30.12  15.829  21.17  23.99  46.924  13.122  
28.86 36.587  19.18


 > head(cpm(currentDiff$counts))

LT1C     LT2C   LT3C   ST2C    ST4C   ST10C   ST12C ST5C    ST7C   
ST8C    ST11C    LT1P    LT2P

hsa-miR-140-5p 215.684 196.8196 237.43 307.42 605.485 285.767 142.576 
288.156 546.467 593.42 153.0286 458.283 167.040

hsa-miR-133a-3p 20.365  13.6880  52.59  49.49   4.284   5.964  33.947 
56.448   3.787  16.07   1.7194  34.058   2.027

hsa-miR-665 8.228  20.4215  37.04  18.78   4.980  20.476  47.525 3.794  
10.529  16.87  13.2864   3.724  26.920

hsa-miR-1250-5p 1.440   0.1104  13.28  11.47   3.481  14.611   3.594 
6.449   2.032   3.64   2.1102  32.786   3.379

hsa-miR-381-3p 13.525  22.7397  86.13  50.28  16.761  11.828  34.346 
184.517  37.866  32.41   0.7816  29.335   8.448

hsa-miR-374b-3p 27.822   7.1751  10.66  31.05  32.344  19.780  26.359 
17.602  34.264  25.84   4.8456  25.975  11.151

LT3P   ST2P   ST10P  ST12P    ST7P    ST8P   ST4P   ST5P ST11P      SB

hsa-miR-140-5p 418.080 330.59 487.258 231.94 532.639 720.513 260.36 
599.79 929.610 342.404

hsa-miR-133a-3p 3.094  17.25   6.952  14.44  49.461  52.070  33.58  
29.44 13.915  43.239

hsa-miR-665 15.792  49.73  22.484  15.42   5.321   3.643   9.68  12.85 
3.657   4.795

hsa-miR-1250-5p 11.345  12.85  22.484  19.22  31.491   2.143  26.72  
52.46 71.982  13.100

hsa-miR-381-3p 12.441  47.70  11.948  16.40  24.338  25.928  98.72  
23.34 34.876  78.687

hsa-miR-374b-3p 31.263  32.12  18.899  21.17  27.216  50.891  16.03  
31.40 39.068  20.121


 > head(cpm(currentDiff$pseudo.counts))

LT1C    LT2C   LT3C   ST2C    ST4C   ST10C  ST12C    ST5C ST7C    
ST8C    ST11C    LT1P    LT2P

hsa-miR-140-5p 215.686 196.824 237.42 307.42 605.506 285.765 142.74 
288.149 546.461 593.414 153.0351 458.284 167.054

hsa-miR-133a-3p 20.362  13.698  52.59  49.49   4.256   5.961  33.80 
56.483   3.793  16.071   1.7286  34.088   2.048

hsa-miR-665 8.221  20.425  37.04  18.79   4.953  20.476  47.03   3.781 
10.532  16.869  13.2858   3.712  26.891

hsa-miR-1250-5p 1.432   0.121  13.29  11.47   3.461  14.617   3.71   
6.449 2.037   3.642   2.1169  32.804   3.399

hsa-miR-381-3p 13.517  22.749  86.14  50.28  16.735  11.826  34.44 
184.584 37.867  32.407   0.7897  29.345   8.461

hsa-miR-374b-3p 27.838   7.186  10.65  31.05  32.352  19.779  26.37  
17.595 34.259  25.835   4.8545  25.973  11.165

LT3P   ST2P   ST10P  ST12P    ST7P    ST8P    ST4P   ST5P ST11P      SB

hsa-miR-140-5p 418.084 330.60 487.258 231.96 532.637 720.512 260.347 
599.80 929.622 342.403

hsa-miR-133a-3p 3.073  17.26   6.948  14.45  49.475  52.067  33.578  
29.45 13.909  43.238

hsa-miR-665 15.786  49.68  22.485  15.42   5.309   3.645   9.673 12.85   
3.649   4.794

hsa-miR-1250-5p 11.334  12.86  22.482  19.23  31.489   2.145  26.717  
52.48 72.001  13.099

hsa-miR-381-3p 12.430  47.70  11.945  16.41  24.330  25.929  98.743  
23.34 34.875  78.686

hsa-miR-374b-3p 31.272  32.12  18.897  21.18  27.212  50.889  16.025  
31.40 39.072  20.120


 > colSums(cpm(currentDiff))

LT1C    LT2C    LT3C    ST2C    ST4C   ST10C   ST12C ST5C    ST7C    
ST8C   ST11C    LT1P    LT2P    LT3P    ST2P

1421292 1064057  981465  889765  960819  921314  985099  991736 1160034 
1144623 1517511  864691 1220229  961164  937648

   ST10P ST12P    ST7P    ST8P    ST4P    ST5P   ST11P      SB

  837525 999688  881438  922050  818447  919173  936499  953165


 > colSums(cpm(currentDiff$counts))

LT1C  LT2C  LT3C  ST2C  ST4C ST10C ST12C  ST5C  ST7C  ST8C ST11C  LT1P  
LT2P  LT3P  ST2P ST10P ST12P  ST7P  ST8P  ST4P

1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 
1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06

  ST5P ST11P    SB

1e+06 1e+06 1e+06


 > colSums(cpm(currentDiff$pseudo.counts))

LT1C  LT2C  LT3C  ST2C  ST4C ST10C ST12C  ST5C  ST7C  ST8C ST11C  LT1P  
LT2P  LT3P  ST2P ST10P ST12P  ST7P  ST8P  ST4P

1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 
1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06

  ST5P ST11P    SB

1e+06 1e+06 1e+06



>  sessionInfo()

R version 3.0.2 (2013-09-25)

Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:

[1] LC_COLLATE=Italian_Italy.1252 LC_CTYPE=Italian_Italy.1252 
LC_MONETARY=Italian_Italy.1252

[4] LC_NUMERIC=C                   LC_TIME=Italian_Italy.1252

attached base packages:

[1] stats     graphics  grDevices utils     datasets  methods base

other attached packages:

[1] xlsx_0.5.5     xlsxjars_0.5.0 rJava_0.9-6    edgeR_3.4.2 limma_3.18.9

loaded via a namespace (and not attached):

[1] tools_3.0.2





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