MINFI missing samples in qcreports and densityplots
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@annabellecongras-12119
Last seen 6.0 years ago

Hello everybody, 

I am using Minfi (1.20.2) to analyse data from Illumina MethylEPIC 850k beadchip. I have 24 samples on 3 arrays. 

I am pretty new to this kinf of analysis, and R in general, so my question may seem really naïve, but I would really appreciate your help. 

When I run qcReport, I have 6 missing samples in the report for DensityPlot and DensityBeanPlot but all samples are present in all others control graphs. I observe the same by running directly densityplot. It seems that the problem come from sampGroups and sampName, because all samples are present when I only run densiPlots without sampName or SampGroup. I don't understant what went wrong... any ideas? Moreover the previous and following steps seem to works fine in the script. 

Here is my code : 

## Set working directory 
setwd(dir="C:/Users/Laure/Documents/iScan/AC166/Minfi_analysis")

## Load packages
require(minfi)
require(limma)
require(IlluminaHumanMethylationEPICmanifest)
require(IlluminaHumanMethylationEPICanno.ilm10b2.hg19)
require(RColorBrewer)

ann850k = getAnnotation(IlluminaHumanMethylationEPICanno.ilm10b2.hg19)

## set directory that contains the idat files
idatdir = "C:/Users/Laure/Documents/iScan/AC166/IDAT" 
list.files(path = idatdir) ##OK found the 3 folder and the sample sheet
list.files(file.path(path=idatdir,"200989060234")) ##OK found 8 green and 8 red

## read the sample sheet that describes the experiment
targets = read.metharray.sheet(idatdir,pattern="samplesheetminfi.csv") 
targets ## OK

## read IDAT files specified in targets file
rgSet = read.metharray.exp(targets = targets)
head(rgSet) ##OK
pheno <- pData(rgSet)
pheno

## calculate the detection p-values
pVals = detectionP(rgSet)
failed.01<- pVals > 0.01
failedProbes <-rownames(failed.01)[rowMeans(failed.01)>0]
sum(rowMeans(failed.01)>0)  # how many probes failed in more thant 0% of samples : 13604
failedProbes

## plot mean detection p-values for all samples
jpeg(filename="Mean_Detection_pVal.jpg")
dotchart(apply(pVals,2,mean),las=2, main="Mean Detection p-values")
dev.off()
#abline(h=0.05,col="red")

## generate a quality control report for the raw data
qcReport(rgSet, sampNames = pheno$SampleName, sampGroups = pheno$Group, maxSamplesPerPage = 24, pdf="qcReport.pdf", controls = c("BISULFITE CONVERSION I", "BISULFITE CONVERSION II", "EXTENSION", "HYBRIDIZATION","NON-POLYMORPHIC", "SPECIFICITY I", "SPECIFICITY II", "TARGET REMOVAL"))
## MISSING 6 SAMPLES!

## Densityplot before normalisation 
densityPlot(rgSet, sampGroups=pheno$Group, add=TRUE, legend = TRUE, main = "Density plots before normalisation") ### MISSING 6 SAMPLES!
densityBeanPlot(rgSet, sampGroups=targets$Group, numPositions = 1000, main = "Bean plots before normalisation")### MISSING 6 SAMPLES!
densityPlot(rgSet, main = "Density plots before normalisation") ## WORKING
> sessionInfo()
R version 3.3.2 (2016-10-31)
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                                  IlluminaHumanMethylationEPICanno.ilm10b2.hg19_0.6.0
 [3] IlluminaHumanMethylationEPICmanifest_0.3.0          limma_3.30.7                                       
 [5] minfi_1.20.2                                        bumphunter_1.14.0                                  
 [7] locfit_1.5-9.1                                      iterators_1.0.8                                    
 [9] foreach_1.4.3                                       Biostrings_2.42.1                                  
[11] XVector_0.14.0                                      SummarizedExperiment_1.4.0                         
[13] GenomicRanges_1.26.2                                GenomeInfoDb_1.10.2                                
[15] IRanges_2.8.1                                       S4Vectors_0.12.1                                   
[17] Biobase_2.34.0                                      BiocGenerics_0.20.0                                

loaded via a namespace (and not attached):
 [1] genefilter_1.56.0        splines_3.3.2            lattice_0.20-34          beanplot_1.2             rtracklayer_1.34.1      
 [6] GenomicFeatures_1.26.2   XML_3.98-1.5             survival_2.39-5          DBI_0.5-1                BiocParallel_1.8.1      
[11] registry_0.3             rngtools_1.2.4           doRNG_1.6                matrixStats_0.51.0       plyr_1.8.4              
[16] pkgmaker_0.22            stringr_1.1.0            zlibbioc_1.20.0          codetools_0.2-15         memoise_1.0.0           
[21] biomaRt_2.30.0           AnnotationDbi_1.36.0     illuminaio_0.16.0        preprocessCore_1.36.0    Rcpp_0.12.8             
[26] xtable_1.8-2             openssl_0.9.6            base64_2.0               annotate_1.52.1          Rsamtools_1.26.1        
[31] digest_0.6.11            stringi_1.1.2            nor1mix_1.2-2            grid_3.3.2               GEOquery_2.40.0         
[36] quadprog_1.5-5           tools_3.3.2              bitops_1.0-6             magrittr_1.5             siggenes_1.48.0         
[41] RCurl_1.95-4.8           RSQLite_1.1-1            MASS_7.3-45              Matrix_1.2-7.1           data.table_1.10.0       
[46] httr_1.2.1               reshape_0.8.6            R6_2.2.0                 mclust_5.2.1             nlme_3.1-128            
[51] GenomicAlignments_1.10.0 multtest_2.30.0 

 

minfi illumina methylationEPIC densityplot • 1.1k views
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@kasper-daniel-hansen-2979
Last seen 4 months ago
United States
Look at the pheno object; perhaps there are missing values? On Fri, Jan 6, 2017 at 2:47 PM, annabelle.congras [bioc] < noreply@bioconductor.org> wrote: > Activity on a post you are following on support.bioconductor.org > > User annabelle.congras <https: support.bioconductor.org="" u="" 12119=""/> wrote Question: > MINFI missing samples in qcreports and densityplots > <https: support.bioconductor.org="" p="" 90855=""/>: > > Hello everybody, > > I am using Minfi (1.20.2) to analyse data from Illumina MethylEPIC 850k > beadchip. I have 24 samples on 3 arrays. > > I am pretty new to this kinf of analysis, and R in general, so my question > may seem really naïve, but I would really appreciate your help. > > When I run qcReport, I have 6 missing samples in the report for > DensityPlot and DensityBeanPlot but all samples are present in all others > control graphs. I observe the same by running directly densityplot. It > seems that the problem come from sampGroups and sampName, because all > samples are present when I only run densiPlots without sampName or > SampGroup. I don't understant what went wrong... any ideas? Moreover the > previous and following steps seem to works fine in the script. > > Here is my code : > > ## Set working directory > setwd(dir="C:/Users/Laure/Documents/iScan/AC166/Minfi_analysis") > > ## Load packages > require(minfi) > require(limma) > require(IlluminaHumanMethylationEPICmanifest) > require(IlluminaHumanMethylationEPICanno.ilm10b2.hg19) > require(RColorBrewer) > > ann850k = getAnnotation(IlluminaHumanMethylationEPICanno.ilm10b2.hg19) > > ## set directory that contains the idat files > idatdir = "C:/Users/Laure/Documents/iScan/AC166/IDAT" > list.files(path = idatdir) ##OK found the 3 folder and the sample sheet > list.files(file.path(path=idatdir,"200989060234")) ##OK found 8 green and 8 red > > ## read the sample sheet that describes the experiment > targets = read.metharray.sheet(idatdir,pattern="samplesheetminfi.csv") > targets ## OK > > ## read IDAT files specified in targets file > rgSet = read.metharray.exp(targets = targets) > head(rgSet) ##OK > pheno <- pData(rgSet) > pheno > > ## calculate the detection p-values > pVals = detectionP(rgSet) > failed.01<- pVals > 0.01 > failedProbes <-rownames(failed.01)[rowMeans(failed.01)>0] > sum(rowMeans(failed.01)>0) # how many probes failed in more thant 0% of samples : 13604 > failedProbes > > ## plot mean detection p-values for all samples > jpeg(filename="Mean_Detection_pVal.jpg") > dotchart(apply(pVals,2,mean),las=2, main="Mean Detection p-values") > dev.off() > #abline(h=0.05,col="red") > > ## generate a quality control report for the raw data > qcReport(rgSet, sampNames = pheno$SampleName, sampGroups = pheno$Group, maxSamplesPerPage = 24, pdf="qcReport.pdf", controls = c("BISULFITE CONVERSION I", "BISULFITE CONVERSION II", "EXTENSION", "HYBRIDIZATION","NON-POLYMORPHIC", "SPECIFICITY I", "SPECIFICITY II", "TARGET REMOVAL")) > ## MISSING 6 SAMPLES! > > ## Densityplot before normalisation > densityPlot(rgSet, sampGroups=pheno$Group, add=TRUE, legend = TRUE, main = "Density plots before normalisation") ### MISSING 6 SAMPLES! > densityBeanPlot(rgSet, sampGroups=targets$Group, numPositions = 1000, main = "Bean plots before normalisation")### MISSING 6 SAMPLES! > densityPlot(rgSet, main = "Density plots before normalisation") ## WORKING > > > sessionInfo() > R version 3.3.2 (2016-10-31) > 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 IlluminaHumanMethylationEPICanno.ilm10b2.hg19_0.6.0 > [3] IlluminaHumanMethylationEPICmanifest_0.3.0 limma_3.30.7 > [5] minfi_1.20.2 bumphunter_1.14.0 > [7] locfit_1.5-9.1 iterators_1.0.8 > [9] foreach_1.4.3 Biostrings_2.42.1 > [11] XVector_0.14.0 SummarizedExperiment_1.4.0 > [13] GenomicRanges_1.26.2 GenomeInfoDb_1.10.2 > [15] IRanges_2.8.1 S4Vectors_0.12.1 > [17] Biobase_2.34.0 BiocGenerics_0.20.0 > > loaded via a namespace (and not attached): > [1] genefilter_1.56.0 splines_3.3.2 lattice_0.20-34 beanplot_1.2 rtracklayer_1.34.1 > [6] GenomicFeatures_1.26.2 XML_3.98-1.5 survival_2.39-5 DBI_0.5-1 BiocParallel_1.8.1 > [11] registry_0.3 rngtools_1.2.4 doRNG_1.6 matrixStats_0.51.0 plyr_1.8.4 > [16] pkgmaker_0.22 stringr_1.1.0 zlibbioc_1.20.0 codetools_0.2-15 memoise_1.0.0 > [21] biomaRt_2.30.0 AnnotationDbi_1.36.0 illuminaio_0.16.0 preprocessCore_1.36.0 Rcpp_0.12.8 > [26] xtable_1.8-2 openssl_0.9.6 base64_2.0 annotate_1.52.1 Rsamtools_1.26.1 > [31] digest_0.6.11 stringi_1.1.2 nor1mix_1.2-2 grid_3.3.2 GEOquery_2.40.0 > [36] quadprog_1.5-5 tools_3.3.2 bitops_1.0-6 magrittr_1.5 siggenes_1.48.0 > [41] RCurl_1.95-4.8 RSQLite_1.1-1 MASS_7.3-45 Matrix_1.2-7.1 data.table_1.10.0 > [46] httr_1.2.1 reshape_0.8.6 R6_2.2.0 mclust_5.2.1 nlme_3.1-128 > [51] GenomicAlignments_1.10.0 multtest_2.30.0 > > > > ------------------------------ > > Post tags: minfi, illumina, methylationEPIC, densityplot > > You may reply via email or visit MINFI missing samples in qcreports and densityplots >
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@annabellecongras-12119
Last seen 6.0 years ago

Hi Kasper, thanks for your answer.

The pheno object is complete. Moreover I have the same issue when I use targets$SampleName instead  pheno$SampleName in qcReport or densityPlots. 

About that object, is this normal that I obtain 2 different output when using pData (data.frame) or phenoData (Formal Call AnnotatedDataFrame)? 

 

 

 

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@annabellecongras-12119
Last seen 6.0 years ago

Hi again, 

Actually if I don't provide any of the sampName and sampGroup options, the densityplots and densitybeanplots in my qcreport are all empty.. does it help understanding the problem?

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