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Question: RNASeq time-course analysis with maSigPro: T.fit error when assessing multiple groups
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gravatar for Ryan Goosen
2.6 years ago by
Switzerland
Ryan Goosen0 wrote:

Dear all, maSigPro authors,

I have a RNASeq time-course experiment with four different clonal populations (C, H1-3, HT; two replicates each) that were each assayed at the same time-points (0h, 4h, 24h, and 72h). I've successfully used maSigPro on each clone's time-course individually to identify significant groups of genes with distinct time-course profiles ("Single Series Time Course Experiment" as referred to in the user manual). However, I'd like to use maSigPro's functionality to compare the results between groups. When I attempt a "Multiple Series Time Course Experiment" with all clones I encounter an error when applying the T.fit method.

The RNASeq time-course data has been normalized and "zero rows" removed. I have setup the design matrix as indicated in the user manual. The order of the rownames of the design matrix is the same as the colnames of the count data matrix.

> str(design)
'data.frame':   40 obs. of  7 variables:
 $ Time      : num  0 0 4 4 24 24 72 72 0 0 ...
 $ Replicates: num  1 1 2 2 3 3 4 4 5 5 ...
 $ C        : num  1 1 1 1 1 1 1 1 0 0 ...
 $ H1        : num  0 0 0 0 0 0 0 0 1 1 ...
 $ H2        : num  0 0 0 0 0 0 0 0 0 0 ...
 $ H3        : num  0 0 0 0 0 0 0 0 0 0 ...
 $ HT       : num  0 0 0 0 0 0 0 0 0 0 ...

 > str(counts)
 num [1:19311, 1:40] 10 0 454 4480 0 ...
 - attr(*, "dimnames")=List of 2
  ..$ : chr [1:19311] "100009600" "100009614" "100017" "100019" ...
  ..$ : chr [1:40] "C_0h.1" "C_0h.3" "C_4h.1" "C_4h.3" ...

> rownames(design) == colnames(counts)
 [1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[16] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
[31] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE

> masigpro.design.matrix = make.design.matrix(design)

> str(masigpro.design.matrix)
List of 3
 $ dis          :'data.frame':  40 obs. of  14 variables:
  ..$ H1vsC   : num [1:40] 0 0 0 0 0 0 0 0 1 1 ...
  ..$ H2vsC   : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ H3vsC   : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ HTvsC  : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ Time     : num [1:40] 0 0 4 4 24 24 72 72 0 0 ...
  ..$ TimexH1  : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ TimexH2  : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ TimexH3  : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ TimexHT : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ Time2    : num [1:40] 0 0 16 16 576 ...
  ..$ Time2xH1 : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ Time2xH2 : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ Time2xH3 : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ Time2xHT: num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
 $ groups.vector: chr [1:14] "H1vsC" "H2vsC" "H3vsC" "HTvsC" ...
 $ edesign      :'data.frame':  40 obs. of  7 variables:
  ..$ Time      : num [1:40] 0 0 4 4 24 24 72 72 0 0 ...
  ..$ Replicates: num [1:40] 1 1 2 2 3 3 4 4 5 5 ...
  ..$ C        : num [1:40] 1 1 1 1 1 1 1 1 0 0 ...
  ..$ H1        : num [1:40] 0 0 0 0 0 0 0 0 1 1 ...
  ..$ H2        : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ H3        : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...
  ..$ HT       : num [1:40] 0 0 0 0 0 0 0 0 0 0 ...

> masigpro.design.matrix$groups.vector
 [1] "H1vsC"  "H2vsC"  "H3vsC"  "HTvsC" "C"      "H1vsC"  "H2vsC"
 [8] "H3vsC"  "HTvsC" "C"      "H1vsC"  "H2vsC"  "H3vsC"  "HTvsC"


> fit <- p.vector(counts, masigpro.design.matrix, Q = 0.05, MT.adjust = "BH", min.obs = 20, counts=TRUE)

...[1] "fitting gene 19300 out of 19311"

> tstep <- T.fit(fit, step.method = "backward", alfa = 0.05)
[1] "fitting gene 100 out of 18237"
[1] "fitting gene 200 out of 18237"
[1] "fitting gene 300 out of 18237"
Error in `colnames<-`(`*tmp*`, value = c("TimexH2", "TimexH3")) :
  'names' attribute [2] must be the same length as the vector [1]

> sessionInfo()
R version 3.2.0 Patched (2015-04-19 r68206)
Platform: x86_64-unknown-linux-gnu (64-bit)
Running under: CentOS release 6.5 (Final)

locale:
 [1] LC_CTYPE=C           LC_NUMERIC=C         LC_TIME=en_US
 [4] LC_COLLATE=en_US     LC_MONETARY=en_US    LC_MESSAGES=en_US
 [7] LC_PAPER=en_US       LC_NAME=C            LC_ADDRESS=C
[10] LC_TELEPHONE=C       LC_MEASUREMENT=en_US LC_IDENTIFICATION=C

attached base packages:
 [1] tcltk     parallel  stats4    stats     graphics  grDevices utils
 [8] datasets  methods   base

other attached packages:
 [1] maSigPro_1.40.0      DynDoc_1.46.0        widgetTools_1.46.0
 [4] BiocInstaller_1.18.1 ggplot2_1.0.1        GO.db_3.1.2
 [7] org.Mm.eg.db_3.1.2   RSQLite_1.0.0        DBI_0.3.1
[10] AnnotationDbi_1.30.0 GenomeInfoDb_1.4.0   IRanges_2.2.0
[13] S4Vectors_0.6.0      Biobase_2.28.0       BiocGenerics_0.14.0
[16] MASS_7.3-40          edgeR_3.10.0         limma_3.24.0

loaded via a namespace (and not attached):
 [1] Rcpp_0.11.5      munsell_0.4.2    colorspace_1.2-6 Mfuzz_2.28.0
 [5] stringr_0.6.2    plyr_1.8.1       tools_3.2.0      grid_3.2.0
 [9] gtable_0.1.2     tkWidgets_1.46.0 digest_0.6.8     reshape2_1.4.1
[13] scales_0.2.4     proto_0.3-10
>


Have I set something up incorrectly? I'd appreciate any insight or assistance that you can provide.

Many thanks,
Ryan Goosen
Department of Biomedicine
University of Basel

ADD COMMENTlink written 2.6 years ago by Ryan Goosen0

Hello Ryan,

I am just curious- did you get any support somewhere or are you even using some other tool and gave up on masigpro? Seems like the developers are not around anymore. I have got some trouble using masigpro as well.

ADD REPLYlink written 2.4 years ago by john40
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