Hi Ramzi, 

My first thought is that something is causing NaNs in the parameter estimates, i.e. maybe one of your genes has the same count for all your samples? 

Try the following: Apply ComBat using the first 20 genes in your your dataset. If ComBat works, add genes (not one at a time, but in sets) until you find the line in your data that makes ComBat fail. If it fails with 20 lines, send me your 20 genes data (offline--no need to send to the entire bioconductor group) and I will take a look.

Hopefully this helps!

Evan



On Nov 14, 2012, at 4:48 PM, Ramzi TEMANNI wrote:

> Hi,
> I have RNA-Seq (host+pathogen) data with this design:
> SampleID	condition	Seq_prot	Exp Date	RNA modif
> 20	1	single-end	1	1
> 82	1	paired-end	2	1
> 86	1	paired-end	3	1
> 21	2	single-end	1	1
> 83	2	paired-end	2	1
> 87	2	paired-end	3	1
> 22	3	single-end	1	1
> 84	3	paired-end	2	1
> 88	3	paired-end	3	1
> 23	4	single-end	1	1
> 85	4	paired-end	2	1
> 89	4	paired-end	3	1
> 90	1	paired-end	4	1
> 91	2	paired-end	4	1
> 92	3	paired-end	4	1
> 93	4	paired-end	4	1
> 94	2	paired-end	4	2
> 95	2	paired-end	4	3
> 
> 
> 
> 
> 
> When I cluster the data, the samples are not clustering by condition and we notice batch effect
> on the Host side: 
> 
> <image.png>
> On the Pathogen side:
> <image.png>
> RNA-Seq data is preprocessed : (Log transformation, quantile normalization, zero variance filtering)
> 
> I used Combat to correct for the batch effect (Exp Date), 
> 
> I run combat on host and pathogen using the following selection of samples:
> 
> 1) All Samples 
> 
> 2) Samples with RNA modif 1
> 
> 3) Sample with exp_date 1/2/3 (without 4)
> 
> 4) Sample with condition 2/3 
> 
> 5) Sample with condition 2/3 AND exp_date 1/2/3 (without 4) 
> 
> with the following command line:
> 
> ComBat(dat=CountTable.IF.LGT, batch=Design$batch, Design$condition, par.prior=T, prior.plots=F)
> For pathogen(selection 4) and host (selection 3 and 5) I get the folowing error:
> 
> Error in while (change > conv) { : missing value where TRUE/FALSE needed
> The low count genes and zero variance genes were removed and there's no missing value in the data, any idea why this error is showing ?
> 
> 
> 
> Thanks in advance,
> 
> Best,
> 
> Ramzi
> 
> 
> 
> 
> 
> 
> > sessionInfo()
> 
> R version 2.15.1 Patched (2012-09-16 r60732)
> 
> Platform: x86_64-apple-darwin9.8.0/x86_64 (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] grid      stats     graphics  grDevices utils     datasets  methods   base     
> 
> other attached packages:
> 
>  [1] DESeq_1.8.3           locfit_1.5-8          Biobase_2.16.0        BiocGenerics_0.2.0    RColorBrewer_1.0-5    gplots_2.11.0        
> 
>  [7] MASS_7.3-22           KernSmooth_2.23-8     caTools_1.13          bitops_1.0-4.1        gdata_2.12.0          gtools_2.7.0         
> 
> [13] sva_3.2.1             mgcv_1.7-21           corpcor_1.6.4         preprocessCore_1.18.0
> 
> loaded via a namespace (and not attached):
> 
>  [1] annotate_1.34.1      AnnotationDbi_1.18.4 DBI_0.2-5            genefilter_1.38.0    geneplotter_1.34.0   IRanges_1.14.4      
> 
>  [7] lattice_0.20-10      Matrix_1.0-9         nlme_3.1-105         RSQLite_0.11.2       splines_2.15.1       stats4_2.15.1       
> 
> [13] survival_2.36-14     tools_2.15.1         XML_3.95-0.1         xtable_1.7-0        
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Mohamed-Ramzi TEMANNI, Ph.D.
> 
> Bioinformatics Research Associate
> 
> Department of Cell Biology and Molecular Genetics
> 
> Maryland Pathogen Research Institute (MPRI)
> 
> 3225 Biosciences Research Bldg
> 
> University of Maryland
> 
> College Park, MD 20742
> 


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