[BioC] problems normalizing in limma
Gordon Smyth
smyth at wehi.edu.au
Thu Oct 13 12:25:57 CEST 2005
The output you give shows that all your intensity matrices (R, G, Rb and
Gb) are one row longer than your annotation data.frame RG2$genes. This
cannot be. The layout information agree with the dimension of RG2$genes.
My guess is that your input gpr files contained a spurious extra row which
is not real data. If so, you need to fix them to remove this spurious row,
or simply remove the last row of all the intensity columns. (Check first
that RG2$R[38977,] is not real data.)
Gordon
>Date: Wed, 12 Oct 2005 18:35:32 -0600
>From: "Anand C.Patel" <acpatel at usa.net>
>Subject: [BioC] problems normalizing in limma
>To: <bioconductor at stat.math.ethz.ch>
>Cc: scrosby at watson.wustl.edu, sbrody at im.wustl.edu
>
>I'm having troubles with some new arrays we're working with.
>
>After some effort, I've managed to read .csv output from PerkinElmer ScanArray
>into an RGList object.
>
>Warning to anyone else -- the .gpr files this program generates do not conform
>strictly to the gpr file format, and will make both limma and marray very
>unhappy.
>
>I have a gal file which appears to correctly identify block, column, row, ID,
>and name, and have even managed to make a SpotTypes file that shows me the
>control probes.
>
>The RGList looks like this (sorry for the large message):
> > RG2
>An object of class "RGList"
>$R
> slide_13295067 slide_13295072 slide_13295071 slide_13295073
>slide_13295075 slide_13295079 slide_13295080 slide_13295203 slide_13295068
>slide_13295214
>[1,] 61323 57928 57404 55737
>61890 58153 63823 53921 57871
>59289
>[2,] 57955 51322 55691 60830
>48322 64418 59731 47373 53971
>54898
>[3,] 1057 3361 1050 1097
>1651 1174 1349 2176 1220
>1471
>[4,] 3185 7716 5754 5925
>5850 8287 8498 6246 3255
>7011
>[5,] 1306 810 1069 533
>978 1485 277 326 1190
>679
> slide_13295879 slide_13295887
>[1,] 58546 58522
>[2,] 59005 61561
>[3,] 1440 1566
>[4,] 13061 12547
>[5,] 24787 648
>38972 more rows ...
>
>$G
> slide_13295067 slide_13295072 slide_13295071 slide_13295073
>slide_13295075 slide_13295079 slide_13295080 slide_13295203 slide_13295068
>slide_13295214
>[1,] 59066 58672 47495 47399
>63671 59502 53084 62982 55238
>52893
>[2,] 52584 54957 39850 50818
>54542 62064 47929 57410 48840
>49985
>[3,] 2443 1444 812 2001
>598 1127 2465 1558 1308
>1148
>[4,] 5249 6511 2926 7372
>4312 5657 10363 8768 2464
>6809
>[5,] 1662 1060 481 954
>936 1071 608 920 512
>438
> slide_13295879 slide_13295887
>[1,] 57486 51338
>[2,] 58792 56861
>[3,] 959 1349
>[4,] 12943 9490
>[5,] 776 994
>38972 more rows ...
>
>$Rb
> slide_13295067 slide_13295072 slide_13295071 slide_13295073
>slide_13295075 slide_13295079 slide_13295080 slide_13295203 slide_13295068
>slide_13295214
>[1,] 172 193 186 208
>220 174 205 237 183
>185
>[2,] 168 191 183 252
>197 214 195 204 202
>259
>[3,] 140 151 145 150
>157 146 151 185 142
>215
>[4,] 148 144 155 153
>147 152 149 190 152
>182
>[5,] 133 140 147 147
>132 143 147 185 144
>147
> slide_13295879 slide_13295887
>[1,] 208 228
>[2,] 210 230
>[3,] 171 152
>[4,] 147 143
>[5,] 152 145
>38972 more rows ...
>
>$Gb
> slide_13295067 slide_13295072 slide_13295071 slide_13295073
>slide_13295075 slide_13295079 slide_13295080 slide_13295203 slide_13295068
>slide_13295214
>[1,] 252 250 261 319
>301 285 355 588 301
>319
>[2,] 247 273 280 300
>279 294 324 568 251
>310
>[3,] 190 197 208 246
>219 187 265 497 223
>249
>[4,] 185 198 219 247
>244 179 263 473 217
>239
>[5,] 177 206 210 242
>278 196 248 501 233
>247
> slide_13295879 slide_13295887
>[1,] 310 270
>[2,] 287 286
>[3,] 237 197
>[4,] 223 222
>[5,] 233 226
>38972 more rows ...
>
>$targets
>[1] "slide_13295067.csv" "slide_13295072.csv" "slide_13295071.csv"
>"slide_13295073.csv" "slide_13295075.csv"
>7 more rows ...
>
>$genes
> Block Column Row ID Name
>Status
>1 1 1 1 mCP000073 gi|21070949|ref|NM_019639.2|_151
>control
>2 1 2 1 mCP000085 gi|21070949|ref|NM_019639.2|_151
>control
>3 1 3 1 mCT000169 NM_008512.90|chr10|-|127557334|127558231_72
>control
>4 1 4 1 mCP000181 gi|31981889|ref|NM_009735.2|_43
>control
>5 1 5 1 mCT000265 NM_011701.9|chr2|+|13576298|13576633_142
>control
>38971 more rows ...
>
>$printer
>$ngrid.r
>[1] 12
>
>$ngrid.c
>[1] 4
>
>$nspot.r
>[1] 28
>
>$nspot.c
>[1] 29
>
>attr(,"class")
>[1] "PrintLayout"
>
>Trying to normalize with either loess or robustspline yields:
>
> > MA <- normalizeWithinArrays(RG2)
>Error in switch(method, loess = { : printer layout information does not match
>M row dimension
>
> > MA <- normalizeWithinArrays(RG2, method="robustspline")
>Loading required package: MASS
>Loading required package: splines
>Error in normalizeRobustSpline(object$M[, j], object$A[, j], layout, df = df,
>:
> (subscript) logical subscript too long
>
>Both suggest that something is amiss with my layout information, but I cannot
>begin to imagine how to diagnose the exact problem.
>
>Help!
>
>Thanks,
>Anand C. Patel, MD
>Washington University School of Medicine
>acpatel at usa.net
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