Cusanovich et al. have prepared a fantastic dataset of transcription factors knocked down with siRNA in a lymphoblastoid cell line. This is a perfect dataset for producing a gold standard form the metaDEGth package

"201 samples were analyzed - 57 transcription factors were knocked down in triplicate in the same cell line, 2 factors were knocked down in 6 replicates each and an additional 18 control knockdowns (siRNA not targeting any known gene) were analyzed"

Cusanovich et al. assayed many siRNA knockdowns of trancription factors in a lymphoblastoid cell line. Each knockdown was performed in triplicate (except for SP1 and IRF5 which were in hexaplet). 18 controls (siRNAs that were not known to bind to a gene product) were also collected. This constitutes an excellent testing and validation set for active regulon detection.

The authors use Illumina HumanHT-12 V4.0 expression beadchips, so a pipeline that makes use of the detection P-value should be employed. Luckily limma solves the problem for us!

Import data

Download the unnormalised zipped expression data directly from GEO

library(data.table)

GSE50588_unnormalised_RAW_DT <- fread(
  cmd = "curl ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE50nnn/GSE50588/suppl/GSE50588_non-normalized.txt.gz | zcat",
  check.names = TRUE)

Build an annotation frame for the experimental system

library(stringr)

samples_GSE50588_metadataDT <- data.table(
  sampleNames = names(GSE50588_unnormalised_RAW_DT) )

samples_GSE50588_metadataDT[, TFknockdown := shift(sampleNames)]
samples_GSE50588_metadataDT[sampleNames %like% "Detect", sampleNames := str_c(TFknockdown,"_DetectionPval")]
samples_GSE50588_metadataDT[!sampleNames %like% "detect", TFknockdown :=  sampleNames]
samples_GSE50588_metadataDT[, TFknockdown := str_remove(TFknockdown, "_\\d(_rep)?")]

samples_GSE50588_metadataDT[sampleNames %like% "NS", TFknockdown := "CONTROL"]

samples_GSE50588_metadataDT[,
                            type := ifelse(str_detect(sampleNames,"Pval"),"pValue","expression"),
                            by = sampleNames]
samples_GSE50588_metadataDT[sampleNames == "ID_REF", type := "ID"]

TF_present <- samples_GSE50588_metadataDT[type == "expression",.N ,by = TFknockdown][order(N)]

Background correct and quantile normalise

We shall make use of the neqc function in the limma package, which is designed for use with Illumina BeadChips; it performs background correction followed by quantile normalisation across all samples, using detection P-values provided by the BeadArray detector.

library(limma)

setnames(GSE50588_unnormalised_RAW_DT, samples_GSE50588_metadataDT$sampleNames)

exprsMat <- GSE50588_unnormalised_RAW_DT[, 
                                    as.matrix(.SD, rownames = ID_REF),
                    .SDcols = samples_GSE50588_metadataDT[type == "expression", sampleNames] ]

detectionPvalueMat <- GSE50588_unnormalised_RAW_DT[,
                                    as.matrix(.SD, rownames = ID_REF),
                    .SDcols = samples_GSE50588_metadataDT[type == "pValue", sampleNames] ]

GSE50588_expObj <- neqc(x = exprsMat,
                        detection.p = detectionPvalueMat)

Map Ilumina probe IDs

GEO provides an annotation file detailing GPL10558, corresponding to the HumanHT-12 v4 Expression BeadChip used in the experiment.

GPL10558_annot_DT <- fread(cmd = "curl ftp://ftp.ncbi.nlm.nih.gov/geo/platforms/GPL10nnn/GPL10558/suppl/GPL10558_HumanHT-12_V4_0_R2_15002873_B.txt.gz | zcat", skip = 8, header = TRUE, fill = TRUE)

GPL10558_annot_DT <- unique(GPL10558_annot_DT[Probe_Id != "",.(Probe_Id, RefSeq_ID, Entrez_Gene_ID, Symbol ,Definition)])

setkey(GPL10558_annot_DT, "Probe_Id")

Fit limma model and estimate differential expression

A linear model fit first requires a design matrix detailing what contrasts can be drawn between which experiments.

assayedTFs <- samples_GSE50588_metadataDT[type == "expression" & TFknockdown != "CONTROL", unique(TFknockdown)]

GSE50588_designMat <- model.matrix(sampleNames ~ 0 + TFknockdown,
                                   samples_GSE50588_metadataDT[type == "expression",.(sampleNames,TFknockdown)])

colnames(GSE50588_designMat) %<>% str_remove("TFknockdown")
row.names(GSE50588_designMat) <- samples_GSE50588_metadataDT[type == "expression", sampleNames]


GSE50588_TFcontrastMat <- makeContrasts(
              contrasts = str_c("CONTROL-",assayedTFs),
              levels = GSE50588_designMat)

We are now ready to build models and compare contrasts (siRNA knockdowns). The plan is to

library(purrr)

GSE50588_lmFit <- lmFit(GSE50588_eSet,
                        design = GSE50588_designMat) %>%
                  contrasts.fit(contrasts = GSE50588_TFcontrastMat) %>%
                  eBayes

# No point having data that we have no regulons to test!
load("../data/TTRUST.RData")
assayedTFs <- intersect(assayedTFs, TTRUST[,.N,by=TF][N >= 5,TF])

TF_siRNA_knockdowns_diffex <- map(
  assayedTFs,
  ~{ diffexDT <- data.table(topTable(GSE50588_lmFit, coef = which(.x==assayedTFs), number = Inf),
                            keep.rownames = TRUE )

    setnames(diffexDT,
             c("rn","logFC","P.Value"),
             c("HumanHT12v4probeID", str_c(.x,c("_logFC","_pValue")) ))

    setkey(diffexDT, "HumanHT12v4probeID")

    return(diffexDT[,.SD,.SDcols = c("HumanHT12v4probeID", str_c(.x,"_logFC") , str_c(.x,"_pValue"))])
  })

TF_siRNA_knockdowns_GSE50588_summary <- Reduce(function(x,y){x[y]}, TF_siRNA_knockdowns_diffex) %>%    
                                .[GPL10558_annot_DT, , on = .(HumanHT12v4probeID == Probe_Id)]

So 40Mb data files are far too big for inclusion into the package. It is still a useful data set, but it will probably just have to be for the paper rather than bundled into the package.



adamsardar/metaDEGth documentation built on Jan. 28, 2020, 12:17 a.m.