Entering edit mode
Hello all,
Following the advice in this post: C: DEXSeq and Independent filtering , I have been using DEXSeq to analyze mouse samples of different biological sex and age. I specified the sex:time interaction in the design formula and the testForDEU() function seems to have progressed without incident. However, when attempting to use the DEXSeqHTML() and or plotDEXSeq() functions to visualize results, I am getting errors. Here's some basic info about my analysis:
> sampleTable
genotype timepoint sex
1071D wt 1m f
1071E wt 1m f
1071G wt 1m f
1071H wt 1m f
1071I wt 1m f
1071P wt 1m m
1071Q wt 1m m
1071X wt 1m m
1072B wt 1m m
1071T wt 1m m
1050B wt 2m f
1050C wt 2m f
1052C wt 2m f
1052I wt 2m f
1053D wt 2m f
1050Q wt 2m m
1052O wt 2m m
1053K wt 2m m
1053L wt 2m m
1053M wt 2m m
1057A wt 4m f
1057B wt 4m f
1057D wt 4m f
1056E wt 4m f
1066D wt 4m f
1048H wt 4m m
1048I wt 4m m
1048K wt 4m m
1056G wt 4m m
1056H wt 4m m
>dxd = DEXSeqDataSetFromHTSeq(
+ countFiles,
+ sampleData=sampleTable,
+ design = ~sample + exon + sex:exon + timepoint:exon + timepoint:sex:exon,
+ flattenedfile = flattenedFile)
>dxd = estimateSizeFactors(dxd)
>dxd = estimateDispersions(dxd, BPPARAM = BPPARAM)
>dxd = testForDEU(dxd, reducedModel <- ~sample + exon + sex:exon + timepoint:exon)
> colors <- brewer.pal(6, "Set2")
> DEXSeqHTML(file = file,path = comparison, dxr1, FDR=0.05 , color = colors)
There were 50 or more warnings (use warnings() to see the first 50)
> warnings()
Warning messages:
1: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
2: In MulticoreParam(workers = 1) :
MulticoreParam not supported on Windows. Use SnowParam instead.
3: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
4: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
5: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
6: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
7: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
8: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
9: closing unused connection 8 (all/files/ENSMUSG00000006782expression.html)
10: closing unused connection 7 (all/files/ENSMUSG00000006390expression.html)
11: closing unused connection 6 (all/files/ENSMUSG00000003402expression.html)
12: closing unused connection 5 (all/files/ENSMUSG00000003355expression.html)
13: closing unused connection 4 (all/files/ENSMUSG00000001786expression.html)
14: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
15: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
16: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
17: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
18: closing unused connection 6 (all/files/ENSMUSG00000017390expression.html)
19: closing unused connection 5 (all/files/ENSMUSG00000013523expression.html)
20: closing unused connection 4 (all/files/ENSMUSG00000008206expression.html)
21: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
22: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
23: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
24: closing unused connection 5 (all/files/ENSMUSG00000020315expression.html)
25: closing unused connection 4 (all/files/ENSMUSG00000019843expression.html)
26: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
27: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
28: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
29: closing unused connection 8 (all/files/ENSMUSG00000021794expression.html)
30: closing unused connection 5 (all/files/ENSMUSG00000021451expression.html)
31: closing unused connection 4 (all/files/ENSMUSG00000020435expression.html)
32: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
33: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
34: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
35: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
36: closing unused connection 8 (all/files/ENSMUSG00000023272expression.html)
37: closing unused connection 5 (all/files/ENSMUSG00000022822expression.html)
38: closing unused connection 4 (all/files/ENSMUSG00000022514expression.html)
39: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
40: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
41: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
42: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
43: closing unused connection 5 (all/files/ENSMUSG00000025408expression.html)
44: closing unused connection 4 (all/files/ENSMUSG00000025372expression.html)
45: In plotDEXSeq(object, geneID = gene, FDR = FDR, lwd = 2, ... :
glm fit failed for gene ENSMUSG00000025733+ENSMUSG00000093593+ENSMUSG00000025735+ENSMUSG00000073434
46: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
47: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
48: In plotDEXSeq(object, geneID = gene, FDR = FDR, lwd = 2, ... :
glm fit failed for gene ENSMUSG00000025733+ENSMUSG00000093593+ENSMUSG00000025735+ENSMUSG00000073434
49: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
50: In plotDEXSeq(object, geneID = gene, FDR = FDR, lwd = 2, ... :
glm fit failed for gene ENSMUSG00000025733+ENSMUSG00000093593+ENSMUSG00000025735+ENSMUSG00000073434
Warning message:
closing unused connection 4 (all/files/ENSMUSG00000090118expression.html)
The plotDEXSeq() function also gives an error:
> plotDEXSeq( dxr1, "ENSMUSG00000028879", expression=FALSE, splicing=TRUE) Error in eval(expr, envir, enclos) : object 'condition' not found In addition: Warning message: In is.na(x) : is.na() applied to non-(list or vector) of type 'NULL'
The plotDEXseq error suggests to me that at least one of the columns in the sampleData *must* be named "condition". The plotDEXSeq function has an argument called "fitExpToVar" which defaults to "condition". The description of this argument is:
"A variable contained in the sample annotation of the DEXSeqDataSet, the expression values will be fitted to this variable using the formula count ~ fitExpToVar * exon."
But I have two factors used in the analysis... "sex" and "timepoint". Should I simply make a 3rd "group" factor which contains all combinations of "sex" and "timepoint" and add it to the sampleData object, or do the referenced columns need to actually be ones used in the design formula?
I have run several DEXSeq analyses with doing pairwise comparisons on these data without incident - so I'm fairly confident that my issue is with the structure of this analysis and not with package installation, etc.
Thanks in advance,
Joseph Bundy
Graduate Student
Florida State University
> sessionInfo() R version 3.2.2 (2015-08-14) Platform: x86_64-w64-mingw32/x64 (64-bit) Running under: Windows 7 x64 (build 7601) Service Pack 1 locale: [1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252 [4] LC_NUMERIC=C LC_TIME=English_United States.1252 attached base packages: [1] stats4 parallel stats graphics grDevices utils datasets methods base other attached packages: [1] RColorBrewer_1.1-2 DEXSeq_1.16.2 DESeq2_1.10.0 RcppArmadillo_0.5.400.2.0 [5] Rcpp_0.12.0 SummarizedExperiment_1.0.1 GenomicRanges_1.22.1 GenomeInfoDb_1.6.1 [9] IRanges_2.4.4 S4Vectors_0.8.3 Biobase_2.30.0 BiocGenerics_0.16.1 [13] BiocParallel_1.4.0 BiocInstaller_1.20.1 RevoUtilsMath_3.2.2
Thanks Alejandro!