I am trying to create a custom made normalization index to implement MAnorm's normalization process with a DESeq2 pipeline for ChIP-seq/ATAC-seq analysis, as it appears the signal/noise ratio is really far off on a couple of samples and its mucking this up. Its a kind of complicated process to paste in here, but basically I'm building this normalizationFactors matrix one column at a time, as I compare it to the average of all samples using the MA method. I have named this matrix "normMatrix" below.
> ColData <- read.table("conds.v2.txt", header=T) > CountData <-read.table("All.merged.ChIP.head.noUn.txt", header=T, row.names=1) > Dds <- DESeqDataSetFromMatrix(countData = CountData, colData = ColData, design = ~ GFP + rep + Lep + Food + GFP*Lep + GFP*Food) > Dds class: DESeqDataSet dim: 60180 16 metadata(1): version assays(1): counts rownames(60180): K27ac_merged_1 K27ac_merged_10 ... K27ac_merged_9998 K27ac_merged_9999 rowData names(0): colnames(16): FasLLL.GFPn.K27ac.rep1 FasLLL.GFPn.K27ac.rep2 ... FedSSS.GFPp.K27ac.rep1 FedSSS.GFPp.K27ac.rep2 colData names(4): Lep GFP rep Food > ncol(normMatrix) [1] 16 > nrow(normMatrix) [1] 60180 > normalizationFactors(Dds) <- normMatrix Error in (function (classes, fdef, mtable) : unable to find an inherited method for function ‘normalizationFactors<-’ for signature ‘"DESeqDataSet", "data.frame"’
I have made sure things are in the appropriate size ranges (both using the mean centering method in the manual and MA alone, which is around 1s anyway), and have replaced zeros with 1s (by inspection they all seem to come from cells with zero read counts, so I'm assuming this won't hurt me). However, I can't see to shake this error. I can run the example code in the ?normalizationFactors man page, so it doesn't seem to a software conflict of some kind. Anyway, I'm a bit baffled, so any help would be greatly appreciated. Thanks.
> sessionInfo() R version 3.3.1 (2016-06-21) Platform: x86_64-apple-darwin13.4.0 (64-bit) Running under: OS X 10.10.5 (Yosemite) 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 stats4 stats graphics grDevices utils datasets methods base other attached packages: [1] MASS_7.3-45 DESeq2_1.12.4 SummarizedExperiment_1.2.3 Biobase_2.32.0 [5] GenomicRanges_1.24.3 GenomeInfoDb_1.8.7 IRanges_2.6.1 S4Vectors_0.10.3 [9] BiocGenerics_0.18.0 loaded via a namespace (and not attached): [1] Rcpp_0.12.7 RColorBrewer_1.1-2 plyr_1.8.4 XVector_0.12.1 tools_3.3.1 [6] zlibbioc_1.18.0 rpart_4.1-10 annotate_1.50.0 RSQLite_1.0.0 gtable_0.2.0 [11] lattice_0.20-33 Matrix_1.2-6 DBI_0.5-1 gridExtra_2.2.1 genefilter_1.54.2 [16] cluster_2.0.4 locfit_1.5-9.1 grid_3.3.1 nnet_7.3-12 data.table_1.9.6 [21] AnnotationDbi_1.34.4 XML_3.98-1.4 survival_2.39-4 BiocParallel_1.6.6 foreign_0.8-66 [26] latticeExtra_0.6-28 Formula_1.2-1 geneplotter_1.50.0 ggplot2_2.1.0 Hmisc_3.17-4 [31] scales_0.4.0 splines_3.3.1 colorspace_1.2-6 xtable_1.8-2 acepack_1.3-3.3 [36] munsell_0.4.3 chron_2.3-47
That did it. Thanks.