Error Running DESeq2

[dpryan]

What happens if you do this instead: des <- ~sampleCondition

[essepf]

DESeq2 --- htseq-count

Hi Michael

Thank you for your suggestion......

hi essepf,

Did you check the length of the count files, as Devon recommended above?

Yes...all files have 38932....

What does sampleCondition look like?

> sampleCondition
[1] pr pr pr wt wt wt
Levels: pr wt


What's your sessionInfo()

> sessionInfo()
R version 3.1.0 (2014-04-10)
Platform: x86_64-apple-darwin13.1.0 (64-bit)

locale:
[1] C

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

other attached packages:
[1] BiocInstaller_1.14.2 gplots_2.14.1 ggplot2_1.0.0 DESeq_1.16.0 lattice_0.20-29 locfit_1.5-9.1 Biobase_2.24.0
[8] DESeq2_1.4.5 RcppArmadillo_0.4.320.0 Rcpp_0.11.2 GenomicRanges_1.16.4 GenomeInfoDb_1.0.2 IRanges_1.22.10 BiocGenerics_0.10.0

loaded via a namespace (and not attached):
[1] AnnotationDbi_1.26.0 DBI_0.2-7 KernSmooth_2.23-12 MASS_7.3-33 RColorBrewer_1.0-5 RSQLite_0.11.4 XML_3.98-1.1 XVector_0.4.0
[9] annotate_1.42.1 bitops_1.0-6 caTools_1.17 colorspace_1.2-4 digest_0.6.4 gdata_2.13.3 genefilter_1.46.1 geneplotter_1.42.0
[17] grid_3.1.0 gtable_0.1.2 gtools_3.4.1 munsell_0.4.2 plyr_1.8.1 proto_0.3-10 reshape2_1.4 scales_0.2.4
[25] splines_3.1.0 stats4_3.1.0 stringr_0.6.2 survival_2.37-7 tools_3.1.0 xtable_1.7-3

[Michael Love]

hi essepf,

Did you check the length of the count files, as Devon recommended above?

What does sampleCondition look like?

What's your sessionInfo()

[essepf]

Hello I have more ou less the same problem:

> ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design = des)
Error in Ops.factor(a$V1, l[[1]]$V1) :
level sets of factors are different

the error is constantly on the factors but I'm not understand why.

I have my ---- sampleCondition=factor and sampleTable=data.frame(sampleName=sampleFiles, fileName=sampleFiles,condition=sampleCondition)

des <- formula(~ condition)

I do not know if you can help with this error.

Thank you very much

[pm2012]

Thanks a lot for help. It was indeed a problem with my count files. I didn't realize I had to redirect the output of HTseq into a different file. I was using file generated with -o option as an input.
I reran the script & was able to generate the correct file (also filtered the last few lines starting with __). The rest of code seems to be working well now.
I also got rid of last colum in sampleTable. It was just one of the many things I was trying to solve my issue.

[dpryan]

After a bit of poking around the source code, it looks like this might happen if there's something wrong with the count files, namely if they're different lengths. From the command line, you might run:

cut -f 1 231-un1-DESeq | sort | uniq -c

If you compare the results of that file to the others the output of uniq -c, which just counts how many unique gene/feature names you have in a file, should be the same...but may not be for you.

BTW, you can get rid of the last column of sampleTable ("stringAsFactors").

[pm2012]

Thanks for your reply.
Here are the contents of sampleTable including the condition. I have 2 conditions with 2 replicates each.

sampleNames sampleFiles condition stringAsFactors
1 231un1 231-un1-DESeq un1 TRUE
2 231un2 231-un2-DESeq un1 TRUE
3 231trt1 231-trt1-DESeq trt2 TRUE
4 231trt2 231-trt2-DESeq trt2 TRUE

Here's the version info:

> sessionInfo()
R version 3.0.2 (2013-09-25)
Platform: x86_64-pc-linux-gnu (64-bit)

locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C

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

other attached packages:
[1] DESeq2_1.2.10 RcppArmadillo_0.4.200.0 Rcpp_0.11.1
[4] GenomicRanges_1.14.4 XVector_0.2.0 IRanges_1.20.7
[7] DESeq_1.14.0 lattice_0.20-24 locfit_1.5-9.1
[10] Biobase_2.22.0 BiocGenerics_0.8.0

loaded via a namespace (and not attached):
[1] annotate_1.40.1 AnnotationDbi_1.24.0 DBI_0.2-7
[4] genefilter_1.44.0 geneplotter_1.40.0 grid_3.0.2
[7] RColorBrewer_1.0-5 RSQLite_0.11.4 splines_3.0.2
[10] stats4_3.0.2 survival_2.37-7 XML_3.98-1.1
[13] xtable_1.7-3

[dpryan]

That's a new one. What are the contents of "sampleTable" and "condition"? Also, which version (just type "sessionInfo()" and post the results)?

[pm2012]

Hi

I am trying to run DESeq2 using the reference manual provided in the bioconductor website. However I am running in the following error after this step: ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory, design= ~ condition) I created my count files using HTseq.

Error msg.

Error in Ops.factor(a$V1, l[[1]]$V1) : level sets of factors are different In addition: Warning message: In is.na(e1) | is.na(e2) : longer object length is not a multiple of shorter object length

Any help is appreciated as I am both R/bioconductor and DESeq2 newbie.