[DESEQ2] VST with rlog + normalization (size factor estimation) for downstream (clustering) analysis.
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john ▴ 110
Last seen 7.1 years ago

Hello guys.

I am using the rlog transformation from DEseq2 (see first figure) and it produces a strange graph. 1th graph are random numbers. 2nd are the raw counts. 3rd are normalized raw counts etc. 

Question1): In the 5th graph- Why are the red dots so close to the x axis? It should more or less look like the first graph. Any ideas on whats causing this?


After some thinking I went on and reduced my data keeping only genes that are expressed in among ALL samples (second figure).

Here rlog does what its supposed to do.





Question2) While we are at it- Is this the correct order for normalization (estimateSizeFactors) + VST(variance stabilizing transformation):


1) rlogTransformation(dds)

2) estimateSizeFactors(dds)

It cant be anything else since rlogTransformation(dds) only takes a DEseqdataset as input with integers. After estimateSizeFactors(dds) and obtaining the counts with counts() the integers are transformed to double. So there is no way I could use rlogTransformation(counts(dds,normalized=T)), right?


Thanks a lot!



For the sake of completeness- the figures are generated with the "vsn" package and the transformations with "DEseq2".

deseq2 normalization variancestabilizingtransformation • 3.7k views
Entering edit mode
Last seen 14 hours ago
United States

Can you send me via email the data and code which produced plot #5 and also your sessionInfo()? If the data is simulated, it could be something funny about the simulation for these rows which doesn't accord with the distributional assumptions. [Edit: see my other answer]

rlog() and varianceStabilizingTransformation() will either use pre-existing size factors or calculate size factors internally if they are not already calculated. Both of these functions only take raw counts, and size factors are used internally in the modeling. The whole point is to use the raw count values for modeling variance.

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Dear Michael,

I did as you said and sent an email to your @gmail.com address. I accidentally included the library("reshape2") - you dont need that, just remove or ignore it.

The data is not simulated. Here is the session info:

> sessionInfo()
R version 3.1.1 (2014-07-10)
Platform: i686-pc-linux-gnu (32-bit)

 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=de_DE.UTF-8        LC_COLLATE=en_US.UTF-8    
 [7] LC_PAPER=de_DE.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            

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

other attached packages:
 [1] gridExtra_0.9.1           reshape2_1.4.1           
 [3] vsn_3.34.0                Biobase_2.26.0           
 [5] ggplot2_1.0.1             DESeq2_1.6.3             
 [7] RcppArmadillo_0.4.650.1.1 Rcpp_0.11.5              
 [9] GenomicRanges_1.18.4      GenomeInfoDb_1.2.4       
[11] IRanges_2.0.1             S4Vectors_0.4.0          
[13] BiocGenerics_0.12.1      

loaded via a namespace (and not attached):
 [1] acepack_1.3-3.3       affy_1.44.0           affyio_1.34.0        
 [4] annotate_1.44.0       AnnotationDbi_1.28.2  base64enc_0.1-2      
 [7] BatchJobs_1.6         BBmisc_1.9            BiocInstaller_1.16.4
[10] BiocParallel_1.0.3    brew_1.0-6            checkmate_1.5.2      
[13] cluster_2.0.1         codetools_0.2-11      colorspace_1.2-6     
[16] DBI_0.3.1             digest_0.6.8          fail_1.2             
[19] foreach_1.4.2         foreign_0.8-63        Formula_1.2-0        
[22] genefilter_1.48.1     geneplotter_1.44.0    gtable_0.1.2         
[25] Hmisc_3.15-0          iterators_1.0.7       KernSmooth_2.23-14   
[28] lattice_0.20-31       latticeExtra_0.6-26   limma_3.22.7         
[31] locfit_1.5-9.1        MASS_7.3-40           munsell_0.4.2        
[34] nnet_7.3-9            plyr_1.8.1            preprocessCore_1.28.0
[37] proto_0.3-10          RColorBrewer_1.1-2    rpart_4.1-9          
[40] RSQLite_1.0.0         scales_0.2.4          sendmailR_1.2-1      
[43] splines_3.1.1         stringr_0.6.2         survival_2.38-1      
[46] tools_3.1.1           XML_3.98-1.1          xtable_1.7-4         
[49] XVector_0.6.0         zlibbioc_1.12.0

Entering edit mode
Last seen 14 hours ago
United States

hi John

I took a look and then realized that those are just genes with all 0 counts in the original DESeqDataSet. 

In ?rlog we say:

To avoid returning matrices with NA                     
values, in the case of a row of all zeros, the rlog transformation                              
returns zeros (essentially adding a pseudocount of 1 only to these                              

These rows can be removed beforehand with:

dds <- dds[rowSums(counts(dds)) > 0, ]


Entering edit mode

Wow Michael you are fast- do you ever sleep?

Alright it was my assumption as well that the 0 genes are responsible.

Nice that we have clarified this!

Entering edit mode
minie • 0
Last seen 4.7 years ago

Hello !

I am facing a problem while generating the plot , similar to what @John was doing.But the problem is I am getting an error with ylim i.e.

Error: Unknown parameters: ylim
Execution halted

I dont know what is going wrong were.

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