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Old 02-28-2014, 12:05 PM   #1
spujr
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Default DESeq2 Problems with MA-Plot

Hello,

I am getting an error when I try to do MA-Plot function in DESeq2. My data input is a simple file of the following headers, "GeneName", "Treat1Rep1_RawCounts", Treat1Rep2_RawCounts"....."Treat4Rep2_RawCounts", "Treat4Rep3_RawCounts". I have 4 conditions, 3 reps each, so 12 different raw counts for each gene ID.

I can import the data in fine and get to the results of the DESeq function. Here is my script below:

> countsTable<-read.delim("merged_raw_counts.txt",header=T)
> rownames(countsTable)<-countsTable$Name
> countsTable<-countsTable[,-1]
> head(countsTable)
T0R1 T0R2 T0R3 T0S1 T0S2 T0S3 T1R1 T1R2 T1R3 T1S1 T1S2 T1S3
gene01g00010 0 5 2 3 4 6 4 1 0 7 1 1
gene01g00020 1 0 12 0 6 2 3 3 1 2 0 0
gene01g00030 0 0 0 0 0 0 0 0 0 1 0 0
gene01g00040 0 0 0 0 0 0 0 0 0 0 0 0
gene01g00050 0 0 0 1 0 0 0 0 0 0 0 0
gene01g00060 13 99 77 109 76 82 82 140 115 157 72 118
>countData<-matrix(countsTable)
>colData<-data.frame(condition=factor(c("T0R","T0R","T0R","T0S","T0S","T0S","T1R","T1R","T1R","T1S","T1S","T1S")))
>dds<-DESeqDataSetFromMatrix(countData=countsTable,colData,formula(~condition))
Usage note: the following factors have 3 or more levels:

condition

For DESeq2 versions < 1.3, if you plan on extracting results for
these factors, we recommend using betaPrior=FALSE as an argument
when calling DESeq().
As currently implemented in version 1.2, the log2 fold changes can
vary if the base level is changed, when extracting results for a
factor with 3 or more levels. A solution will be implemented in
version 1.3 which allows for the use of a beta prior and symmetric
log2 fold change estimates regardless of the selection of base level.
>colData(dds)$condition<-factor(colData(dds)$condition,levels=c("T0R","T0S","T1R","T1S"))
> dds
class: DESeqDataSet
dim: 34903 12
exptData(0):
assays(1): counts
rownames(34903): gene01g00010 gene01g00020 ... gene00g99390 gene00g99400
rowData metadata column names(0):
colnames(12): 1 2 ... 11 12
colData names(1): condition
> dds<-DESeq(dds)
> res<-results(dds)
> res<-res[order(res$padj),]
> head(res)
DataFrame with 6 rows and 6 columns
baseMean log2FoldChange lfcSE stat pvalue padj
<numeric> <numeric> <numeric> <numeric> <numeric> <numeric>
gene00g04520 13076.372 5.050936 0.17241390 29.29541 1.186570e-188 3.046400e-184
gene03g01180 9750.315 3.897496 0.13548892 28.76616 5.687327e-182 7.300822e-178
gene00g09910 4133.619 2.299451 0.08051913 28.55783 2.245862e-179 1.869130e-175
gene00g48700 1329.461 -2.640605 0.09249464 -28.54874 2.912098e-179 1.869130e-175
gene04g12160 12921.588 3.599922 0.12872185 27.96667 4.133827e-172 2.122638e-168
gene00g46890 25040.714 4.235810 0.15466115 27.38768 3.845523e-165 1.645499e-161

Up to this point I don't have any problems. However when I do:
> plotMA(dds,ylim=c(-2,2),main="DESeq2")

I get:
Error in plotMA(dds, ylim = c(-2, 2), main = "DESeq2") :
'x' must be a data frame with columns named 'baseMean', 'log2FoldChange'.

My apologies if I posted too much background info. I'd figured my error is in something I did in early steps.

Thanks for any help on this.

-Will
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Old 02-28-2014, 12:59 PM   #2
dpryan
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Code:
plotMA(res, ylim=c(-2,2),main="DESeq2")
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Old 02-28-2014, 01:42 PM   #3
Michael Love
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hi Will,

can you include the sessionInfo() output?

In Bioc 2.13, the plotMA function became a generic defined in the geneplotter package for data.frame, and we transitioned plotMA to an S4 method in DESeq2 which is defined for DESeqDataSet objects (and in DESeq2 version >= 1.3 it also defined for the object returned by results(), a DESeqResults object).

I think if you update the geneplotter package, it might solve your error:

biocLite("geneplotter")
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Old 02-28-2014, 06:21 PM   #4
bpgoll
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Quote:
Originally Posted by spujr View Post
Hello,



My apologies if I posted too much background info. I'd figured my error is in something I did in early steps.

Thanks for any help on this.

-Will
Hopefully anyone can help this
So sorry can't
Good luck sir
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Old 03-04-2014, 11:13 AM   #5
spujr
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Hi,

Thanks for your responses. I thought I updated everything but found out this wasn't the case. Once I followed Michael's suggestion the problem was resolved.

Thanks again!
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Old 03-25-2014, 11:44 AM   #6
cacti
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I am also having a problem with the plotMA function in DESeq2 1.3

First I got this error message:
> plotMA(res, main="DESeq2", ylim=c(-2,2))
Error in as.vector(data) :
no method for coercing this S4 class to a vector

So I updated geneplotter, and I got this error message:

> plotMA(res,ylim=c(-2,2),main="DESeq2")
Error in plotMA(res, ylim = c(-2, 2), main = "DESeq2") :
Error from the generic function 'plotMA' defined in package 'BiocGenerics': no S4 method definition for argument 'res' of class 'DataFrame' was found. Did you perhaps mean calling the function 'plotMA' from another package, e.g. 'limma'? In that case, please use the syntax 'limma:lotMA'.
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Old 03-25-2014, 11:59 AM   #7
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hi,

These function definition problems are all due to package versions (either out of date packages, or using syntax from the vignettes of newer versions with older versions of software), so I can't provide much help unless you include the full output of sessionInfo() with your question.
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Old 03-26-2014, 09:27 AM   #8
geneart
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Hello all,
This seems to be my problem as well ! I have tried plotMA on DESeq and DESeq2 but I get this same error as mentioned by cacti: Here is my session info . hopefully there is something someone can figure out for me! I am desperately trying to ge tthis plot! I plotted plotDE and it works fine . JUst the plotMA is the problem. Thanks in advance !BTW I checked on the function in DESeq2 vignette and it seems to be what I used , no changes.
> sessionInfo()
R version 3.0.1 (2013-05-16)
Platform: i386-w64-mingw32/i386 (32-bit)

locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.1252

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

other attached packages:
[1] DESeq2_1.2.10 RcppArmadillo_0.4.100.2.1
[3] Rcpp_0.11.1 GenomicRanges_1.14.4
[5] XVector_0.2.0 IRanges_1.20.7
[7] DESeq_1.14.0 lattice_0.20-15
[9] locfit_1.5-9.1 Biobase_2.22.0
[11] BiocGenerics_0.8.0 BiocInstaller_1.12.0

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_3.0.1
[7] RColorBrewer_1.0-5 RSQLite_0.11.4 splines_3.0.1
[10] stats4_3.0.1 survival_2.37-7 tools_3.0.1
[13] XML_3.98-1.1 xtable_1.7-3
I also loaded my previous workspace and updated geneplotter and this is what I got:
> plotMA(dseq,pvalCutoff=.05,ylim=c(-2,2))
Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘extractROWS’ for signature ‘"list"’

Thanks very much
geneart.
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Old 03-26-2014, 09:59 AM   #9
cacti
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Hi Michael, I just started a new session and tried to load DESeq2 1.3.59 but I got this error:

> library("DESeq2")
Error in dyn.load(file, DLLpath = DLLpath, ...) :
unable to load shared object '/Library/Frameworks/R.framework/Versions/3.0/Resources/library/DESeq2/libs/DESeq2.so':
dlopen(/Library/Frameworks/R.framework/Versions/3.0/Resources/library/DESeq2/libs/DESeq2.so, 6): Library not loaded: /Library/Frameworks/R.framework/Versions/3.1/Resources/lib/libRlapack.dylib
Referenced from: /Library/Frameworks/R.framework/Versions/3.0/Resources/library/DESeq2/libs/DESeq2.so
Reason: Incompatible library version: DESeq2.so requires version 3.1.0 or later, but libRlapack.dylib provides version 3.0.0
In addition: Warning message:
package ‘DESeq2’ was built under R version 3.1.0
Error: package or namespace load failed for ‘DESeq2’

Here is my session info:

> sessionInfo()
R version 3.0.3 (2014-03-06)
Platform: x86_64-apple-darwin10.8.0 (64-bit)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

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

other attached packages:
[1] limma_3.18.13 gplots_2.12.1 goseq_1.14.0
[4] geneLenDataBase_0.99.12 BiasedUrn_1.06.1 geneplotter_1.40.0
[7] annotate_1.40.1 AnnotationDbi_1.24.0 lattice_0.20-27
[10] Biobase_2.22.0 gdata_2.13.2 biomaRt_2.18.0
[13] BiocInstaller_1.12.0 RcppArmadillo_0.4.100.2.1 Rcpp_0.11.1
[16] GenomicRanges_1.14.4 XVector_0.2.0 IRanges_1.20.7
[19] BiocGenerics_0.8.0

loaded via a namespace (and not attached):
[1] Biostrings_2.30.1 bitops_1.0-6 BSgenome_1.30.0
[4] caTools_1.16 DBI_0.2-7 genefilter_1.44.0
[7] GenomicFeatures_1.14.5 grid_3.0.3 gtools_3.3.1
[10] KernSmooth_2.23-10 locfit_1.5-9.1 Matrix_1.1-2
[13] mgcv_1.7-28 nlme_3.1-113 RColorBrewer_1.0-5
[16] RCurl_1.95-4.1 Rsamtools_1.14.3 RSQLite_0.11.4
[19] rtracklayer_1.22.6 splines_3.0.3 stats4_3.0.3
[22] survival_2.37-7 tools_3.0.3 XML_3.95-0.2
[25] xtable_1.7-3 zlibbioc_1.8.0


This information may also be helpful. I received this message when installing limma...
> library("limma", lib.loc="/Library/Frameworks/R.framework/Versions/3.0/Resources/library")

Attaching package: ‘limma’

The following object is masked from ‘package:geneplotter’:

plotMA

The following object is masked from ‘package:BiocGenerics’:

plotMA
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Old 03-26-2014, 10:40 AM   #10
Michael Love
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hi geneart,

You are using DESeq2 v1.2, in which plotMA() can only take a DESeqDataSet object. so this should work:

plotMA(dds)

There's always a bit of confusion because google might direct our users to the development vignettes online, and then ending up with code for the wrong version. It's preferable to load the man pages and vignettes within R, to make sure you get the proper documentation:

?plotMA
vignettes("DESeq2")

plotMA() taking the object returned by results() was a switch we made in the devel branch (DESeq2 v1.3), which makes more sense IMO than taking the DESeqDataSet but i didn't realize this until after 1.2 was released.


hi cacti,

You are using the devel version of DESeq2 which was built with R-devel (R version 3.1.0), but it looks like you are running R version 3.0.3. The devel version of our package will be released as DESeq2 v1.4 on April 14.

i'd suggest continuing to use the version of the package which is installed by biocLite("DESeq2"), and if this is version 1.2 then you can provide the DESeqDataSet to plotMA:

plotMA(dds)

Mike
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Old 03-26-2014, 12:02 PM   #11
cacti
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Thanks Mike, I was just excited to try the new version of DESeq2 as I am analyzing a factor with three levels. I had used betaPrior=False when calling DESeq() but I'm curious to see if my results differ using the new version.
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Old 03-26-2014, 12:13 PM   #12
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EDIT: Bioconductor packages should be installed following this advice:
http://bioconductor.org/install/


ah, I see. Yes, we like to have devel testers as well, because we shouldn't push changes to the release that aren't show-stopping bugs. Without devel testers, we should wait 6 months to push subtle improvements.

The p-values shouldn't change much with or without LFC shrinkage, but the effects should be more interpretable, i.e., large absolute LFC is likely meaningful.

Last edited by Michael Love; 07-30-2014 at 05:31 AM. Reason: dont recommend own install
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Old 03-26-2014, 02:06 PM   #13
geneart
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Default DESeq2 plotMA()

Thanks very much Mike !
Will definitely try out what you said and will update soon
Regards
geneart.
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Old 04-05-2014, 04:51 AM   #14
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Hi!

I've got a similar problem with plotMA in DESeq2. Here is my session info:

R version 3.0.2 (2013-09-25)
Platform: i386-w64-mingw32/i386 (32-bit)

locale:
[1] LC_COLLATE=Spanish_Spain.1252 LC_CTYPE=Spanish_Spain.1252 LC_MONETARY=Spanish_Spain.1252
[4] LC_NUMERIC=C LC_TIME=Spanish_Spain.1252

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

other attached packages:
[1] BiocInstaller_1.12.0 VennDiagram_1.6.5 NOISeq_2.6.0
[4] TCC_1.2.0 ROC_1.38.0 baySeq_1.16.0
[7] DESeq_1.14.0 lattice_0.20-29 locfit_1.5-9.1
[10] Biobase_2.22.0 DESeq2_1.2.10 RcppArmadillo_0.4.100.2.1
[13] Rcpp_0.11.1 GenomicRanges_1.14.4 XVector_0.2.0
[16] IRanges_1.20.7 BiocGenerics_0.8.0 edgeR_3.4.2
[19] limma_3.18.13

loaded via a namespace (and not attached):
[1] annotate_1.40.1 AnnotationDbi_1.24.0 DBI_0.2-7 EBSeq_1.2.0
[5] genefilter_1.44.0 geneplotter_1.40.0 RColorBrewer_1.0-5 RSQLite_0.11.4
[9] samr_2.0 stats4_3.0.2 survival_2.37-7 tools_3.0.2
[13] XML_3.98-1.1 xtable_1.7-3


My DESeq2 script is like this:

index.groups <- as.factor( "A","B")))
coldat=DataFrame(grp=factor(index.groups), each=1)
dds <- DESeqDataSetFromMatrix(raw_filter, colData=coldat, design =~grp)
dds <- DESeq(dds)
deseq2.res <- results(dds)
deseq2.res <- deseq2.res[order(deseq2.res$padj<0.05),]
deseq2.fc=deseq2.res$log2FoldChange
names(deseq2.fc)=rownames(deseq2.res)
exp.fc=deseq2.fc
out.suffix="deseq2"
head(exp.fc)
head(deseq2.res)
resultsNames(dds)

So far it works fine, but when I'm using plotMA(dds) appears the following error message:

Error in plotMA(dds) :
'x' must be a data frame with columns named 'baseMean', 'log2FoldChange'.


(I think that all my packages are updated)
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Old 04-05-2014, 05:47 AM   #15
Michael Love
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Hi Beth,

I think it may be that geneplotter or another package is masking plotMA because it was loaded after.

Try:

Code:
DESeq2::plotMA(dds)
(this was the reason why we made plotMA a method in Bioc 2.14 which we could dispatch on different classes of objects)

Last edited by Michael Love; 04-05-2014 at 05:58 AM.
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Old 04-14-2014, 03:34 AM   #16
geneart
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Update on mike's tip on plotMA, it worked ! i used ;
DESeq:: plotMA(res)
or DESeq2:: plotMA(your requirements)

I have another question. I am working with 4 animals as my samples with absolutely NO replicates !!! It is downright expensive to have replicate conditions for the animals and also during the process of library preparation , replicates were not made due to cost of running NGS on all of them and so on....but anyways, right now what I have is what I have My problem is this: I have used DESeq2 to perform the DE analysis as indicated in part one below and DESeq to perform DE analysis as indicated in part 2. For DESeq I am using per sample across its two conditions for evaluation.
For my first part I have used all four samples (treated as replicates) for both untreated and treated condition> basically I have sample1,2,3,4 fed across both untreated and treated into DESeq2. I generated heatmaps of highly expressed genes. However I have only 3 genes differentially expressed with a Padj value of less than 0.05.
Part 2: I am using DESeq per sample using its own untreated vs treated. To indicate I have no replicates I have used
cds<-estimateDispersions(cds,method="blind",sharingMode="fit-only",fitType="local"). I get a result fo rbinom test with a lot of NA !!!!!downstream
however when I do a :
> cdsBlind = estimateDispersions( cds, method="blind" )
> vsd = varianceStabilizingTransformation( cdsBlind )
Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘normalizationFactors’ for signature ‘"CountDataSet"’
I get the above error...........why is that? I had normalized my counts and used that to perform this step . However I still get this error: plz see below;
> a<-counts(cds,normalized=T)
cds3<-estimateDispersions(a,method="blind",sharingMode="fit-only",fitType="local")
Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘estimateDispersions’ for signature ‘"matrix"’
> cdsBlind = estimateDispersions( a, method="blind" )
Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘estimateDispersions’ for signature ‘"matrix"’

Please could u suggest how I can get to get a top highly expressed gene list per sample WITHOUT REPLICATES in DESeq ? Why am I getting this error? I poked a little bit into this googling and I am confused. My "a" is normalized counts.

Also can I compare fold2change values obtained from results of nbinom using DESeq to the fold2change I get from results dseq<-DESeq(dds) in DESeq2?
I appreciate any input for such a condition where one is working without any replicates !!! Thanks very much in advance !

Last edited by geneart; 04-14-2014 at 03:41 AM.
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Old 04-14-2014, 04:38 PM   #17
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hi geneart,

I wouldn't mix DESeq (old package) and DESeq2. The analysis without replicates will work automatically in DESeq2 using the same principles as before (samples are treated as replicates for estimation of dispersion).

from ?estimateDispersions:

Quote:
estimateDispersions checks for the case of an analysis with as many samples as the number
of coefficients to fit, and will temporarily substitute a design formula ~ 1 for the purposes of
dispersion estimation. This treats the samples as replicates for the purpose of dispersion estimation.
As mentioned in the DESeq paper: "While one may not want to draw strong conclusions from such
an analysis, it may still be useful for exploration and hypothesis generation."
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Old 04-14-2014, 06:29 PM   #18
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Thanks Mike for the input. Appreciate it very much !
geneart.
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Old 05-15-2014, 01:09 AM   #19
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Hi Mike,

I was wondering if the release version of DESeq still needs the development version of R. I am still getting the same errors as cacti above with DESeq 1.4.5. Below I've pasted my loading attempt and the error message I received.

> install.packages("~/Downloads/DESeq2_1.4.5.tgz", repos = NULL)
Installing package into ‘/Users/co7/Library/R/3.0/library’
(as ‘lib’ is unspecified)
> library("DESeq2", lib.loc="/Users/co7/Library/R/3.0/library")
Loading required package: GenomicRanges
Loading required package: BiocGenerics
Loading required package: parallel

Attaching package: ‘BiocGenerics’

The following objects are masked from ‘packagearallel’:

clusterApply, clusterApplyLB, clusterCall, clusterEvalQ, clusterExport, clusterMap, parApply, parCapply,
parLapply, parLapplyLB, parRapply, parSapply, parSapplyLB

The following object is masked from ‘package:stats’:

xtabs

The following objects are masked from ‘package:base’:

anyDuplicated, append, as.data.frame, as.vector, cbind, colnames, duplicated, eval, evalq, Filter, Find, get,
intersect, is.unsorted, lapply, Map, mapply, match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
Position, rank, rbind, Reduce, rep.int, rownames, sapply, setdiff, sort, table, tapply, union, unique, unlist

Loading required package: IRanges
Loading required package: XVector
Loading required package: Rcpp
Loading required package: RcppArmadillo
Error in dyn.load(file, DLLpath = DLLpath, ...) :
unable to load shared object '/Users/co7/Library/R/3.0/library/DESeq2/libs/DESeq2.so':
dlopen(/Users/co7/Library/R/3.0/library/DESeq2/libs/DESeq2.so, 6): Library not loaded: /Library/Frameworks/R.framework/Versions/3.1/Resources/lib/libRlapack.dylib
Referenced from: /Users/co7/Library/R/3.0/library/DESeq2/libs/DESeq2.so
Reason: Incompatible library version: DESeq2.so requires version 3.1.0 or later, but libRlapack.dylib provides version 3.0.0
In addition: Warning message:
package ‘DESeq2’ was built under R version 3.1.0
Error: package or namespace load failed for ‘DESeq2’

I have gone back to using version 1.2.10 but I'm hoping to redo my analysis in the new version as soon as a stable release it out. Do you know when that might be? Thank you!

Cheers,
Kwasi
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Old 05-15-2014, 04:08 AM   #20
Michael Love
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hi Kwasi,

If you are getting the same errors as above with plotMA, it is because you have Bioconductor packages out of sync. The best way to fix this is to upgrade all packages to the latest release version.

In the Bioconductor x.y.z version number, if y is even, this means the package is a release version.

So 1.4.5 is the current release version of DESeq2 which you can download if you upgrade to the newest release of R: 3.1.0.

You should update R on your machine, and then update packages with the following advice:

http://bioconductor.org/install/#upd...uctor-packages
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