So this is my script and I have used it in the past. The filtering and then getting my list of DEG. The list contains only pos_controls from affymetrix but no main probes. Which I do not understand because I thought I removed them with the filter step which works on my other 2 datasets. Thanks!

mydir <- "C:\\Users\\hakim\\Documents\\hanane_data"

#listing the files from directory using special CEL file read function celList <- list.celfiles(mydir, full.names=TRUE)
#reading data from cellist and setting annotation package to approiate one for this microarray rawData <- read.celfiles(celList, pkgname='pd.hugene.2.0.st')
#normalizing the data using RMA algorithm normData <- rma(rawData, target="core")
#retreaving feature data featureData(normData) <- getNetAffx(normData, "transcript")

#the respective experimental groups of your data group <- factorcrep.int(0,4)),rep.int(1,4),rep.int(2,4)
                  ,rep.int(3,4),rep.int(4,4),rep.int(5,4)),rep.int(6,4),rep.int(7,4)))
#design and contrast matrix of the data design <- model.matrix(~ 0 + group) colnames(design) <- c("ID01","I01","ID005","I005", "ID3","I3","CtlID","CtlI") contrast <- makeContrasts( "ID01-ID005","ID01-ID3","ID005-ID3","ID01-CtlID","ID005-CtlID","ID3-CtlID",
                           "I01-I005","I01-I3","I005-I3","I01-CtlI","I005-CtlI","I3-CtlI",
                           "ID01-I01","ID005-I005","ID3-I3","CtlI-CtlID",
                           levels= design ) eset <- getMainProbes(normData) normData.filtered <- nsFilter(eset, require.entrez = FALSE,
                              remove.dupEntrez = FALSE)

normfit <-eBayes( contrasts.fit( lmFit(normData.filtered$eset, design), contrast) )

#getting the list of probes probeset.list <-topTable(normfit,coef="ID01-ID3",number=100000, adjust="BH", lfc=1)

R version 3.2.3 (2015-12-10) Platform: x86_64-w64-mingw32/x64 (64-bit) Running under: Windows >= 8 x64 (build 9200)

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

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

other attached packages:  [1] hugenedeg_1.0                        affycoretools_1.42.0                 hugene20sttranscriptcluster.db_8.4.0  [4] org.Hs.eg.db_3.2.3           annotate_1.48.0                      XML_3.98-1.3                      [7] AnnotationDbi_1.32.3                 genefilter_1.52.1             pd.hugene.2.0.st_3.14.1              [10] oligo_1.34.2                 Biostrings_2.38.4                    XVector_0.10.0                    [13] IRanges_2.4.7                        S4Vectors_0.8.11             oligoClasses_1.32.0                  [16] limma_3.26.8                 Biobase_2.30.0                       BiocGenerics_0.16.1               [19] RSQLite_1.0.0                        DBI_0.3.1                   


loaded via a namespace (and not attached):  [1] Category_2.36.0        bitops_1.0-6               RColorBrewer_1.1-2         GenomeInfoDb_1.6.3          [5] gcrma_2.42.0               tools_3.2.3 affyio_1.40.0              KernSmooth_2.23-15          [9] rpart_4.1-10               Hmisc_3.17-2               colorspace_1.2-6 nnet_7.3-12                [13] gridExtra_2.0.0            GGally_1.0.1               DESeq2_1.10.1              bit_1.1-12       [17] preprocessCore_1.32.0      graph_1.48.0               rtracklayer_1.30.2         ggbio_1.18.5               [21] caTools_1.17.1             scales_0.3.0               affy_1.48.0      RBGL_1.46.0                [25] stringr_1.0.0              Rsamtools_1.22.0           foreign_0.8-66             R.utils_2.2.0    [29] AnnotationForge_1.12.2     dichromat_2.0-0            BSgenome_1.38.0            PFAM.db_3.2.2              [33] BiocInstaller_1.20.1       GOstats_2.36.0             hwriter_1.3.2    gtools_3.5.0               [37] BiocParallel_1.4.3         R.oo_1.20.0 acepack_1.3-3.3            VariantAnnotation_1.16.4   [41] RCurl_1.95-4.7             magrittr_1.5               GO.db_3.2.2      Formula_1.2-1              [45] futile.logger_1.4.1        Matrix_1.2-3               Rcpp_0.12.3                munsell_0.4.3    [49] R.methodsS3_1.7.1          stringi_1.0-1              edgeR_3.12.0               SummarizedExperiment_1.0.2 [53] zlibbioc_1.16.0            gplots_2.17.0              plyr_1.8.3       grid_3.2.3                 [57] affxparser_1.42.0          gdata_2.17.0               ReportingTools_2.10.0      lattice_0.20-33  [61] splines_3.2.3              GenomicFeatures_1.22.13    locfit_1.5-9.1             knitr_1.12.3               [65] GenomicRanges_1.22.4       geneplotter_1.48.0         reshape2_1.4.1   codetools_0.2-14           [69] biomaRt_2.26.1             futile.options_1.0.0       RcppArmadillo_0.6.500.4.0  biovizBase_1.18.0          [73] latticeExtra_0.6-28        lambda.r_1.1.7             foreach_1.4.3              gtable_0.1.2     [77] reshape_0.8.5              ggplot2_2.0.0              xtable_1.8-2               ff_2.2-13                  [81] survival_2.38-3            OrganismDbi_1.12.1         iterators_1.0.8  GenomicAlignments_1.6.3    [85] cluster_2.0.3              GSEABase_1.32.0