I am using Limma for downstream analysis of mass spectrometry proteomic data.
I am comparing differential protein expression between healthy controls (n= 40) and two disease subtypes (disease_1 n= 59, disease_2 n= 41). I have an 'intensities' matrix with the log2 transformed LFQ expression for 1300 proteins in rows with individual samples in columns. I also have a patient variables matrix with the columns including sample name, disease subtype, batch, age & sex. I wish to adjust for batch (A-E), as well as sex and age.
I read the Limma user guide and searched the Bioconductor support pages- both of which I found very helpful.
From my understanding, it is most correct to include batch adjustment (and other covariants?) in the linear model. Therefore I have designed my code as outlined below.
My question: Is it possible to extract a fitted expression matrix from the lmfit function in limma (post batch adjustment) which can be used for PCA?
Or is the only possible to get a fitted expression matrix for PCA by independently using the remove::batch function? I have also included this code.
design<-model.matrix(~patientvar$Subtype+patientvar$Batch+ patientvar$Sex +patientvar$Age) fit<-lmFit(intensities,design) fit2 <- eBayes(fit) topTable(fit2, coef=2) topTable(fit2, coef=3) design0 = model.matrix(~patientvar$Subtype) rembatch_intensities <- removeBatchEffect(intensities, batch=patientvar$Batch, batch2 = patientvar$Sex, covariates = patientvar$Age, design = design0) > sessioninfo() setting value version R version 4.3.0 (2023-04-21 ucrt) os Windows 11 x64 (build 22621) system x86_64, mingw32 ui RStudio language (EN) collate English_Ireland.utf8 ctype English_Ireland.utf8 tz America/Los_Angeles date 2023-05-26 rstudio 2023.03.1+446 Cherry Blossom (desktop) pandoc 2.19.2 @ C:/Program Files/RStudio/resources/app/bin/quarto/bin/tools/ (via rmarkdown) package * version date (UTC) lib source BiocManager 1.30.20 2023-02-24  CRAN (R 4.3.0) cli 3.6.1 2023-03-23  CRAN (R 4.3.0) digest 0.6.31 2022-12-11  CRAN (R 4.3.0) evaluate 0.20 2023-01-17  CRAN (R 4.3.0) fastmap 1.1.1 2023-02-24  CRAN (R 4.3.0) htmltools 0.5.5 2023-03-23  CRAN (R 4.3.0) knitr 1.42 2023-01-25  CRAN (R 4.3.0) limma * 3.56.0 2023-04-25  Bioconductor rlang 1.1.1 2023-04-28  CRAN (R 4.3.0) rmarkdown 2.21 2023-03-26  CRAN (R 4.3.0) rstudioapi 0.14 2022-08-22  CRAN (R 4.3.0) sessioninfo 1.2.2 2021-12-06  CRAN (R 4.3.0) xfun 0.39 2023-04-20  CRAN (R 4.3.0) yaml 2.3.7 2023-01-23  CRAN (R 4.3.0)  C:/Users/sarah/AppData/Local/R/win-library/4.3  C:/Program Files/R/R-4.3.0/library