inst/doc/Crispr_example_workflow.R

In gCrisprTools: Suite of Functions for Pooled Crispr Screen QC and Analysis

## ---- eval = FALSE------------------------------------------------------------
#  library(Biobase)
#  library(limma)
#  library(gCrisprTools)
#  
#  data("es", package = "gCrisprTools")
#  data("ann", package = "gCrisprTools")
#  data("aln", package = "gCrisprTools")

## ---- eval = FALSE------------------------------------------------------------
#  sk <- relevel(as.factor(pData(es)$TREATMENT_NAME), "ControlReference")
#  names(sk) <- row.names(pData(es))

## ---- eval = FALSE------------------------------------------------------------
#  design <- model.matrix(~ 0 + REPLICATE_POOL + TREATMENT_NAME, pData(es))
#  colnames(design) <- gsub('TREATMENT_NAME', '', colnames(design))
#  contrasts <-makeContrasts(DeathExpansion - ControlExpansion, levels = design)

## ---- eval = FALSE------------------------------------------------------------
#  es <- ct.filterReads(es, trim = 1000, sampleKey = sk)

## ---- eval = FALSE------------------------------------------------------------
#  es <- ct.normalizeGuides(es, method = "scale", plot.it = TRUE) #See man page for other options
#  vm <- voom(exprs(es), design)
#  
#  fit <- lmFit(vm, design)
#  fit <- contrasts.fit(fit, contrasts)
#  fit <- eBayes(fit)

## ---- eval = FALSE------------------------------------------------------------
#  ann <- ct.prepareAnnotation(ann, fit, controls = "NoTarget")

## ---- eval = FALSE------------------------------------------------------------
#  resultsDF <-
#    ct.generateResults(
#      fit,
#      annotation = ann,
#      RRAalphaCutoff = 0.1,
#      permutations = 1000,
#      scoring = "combined",
#      permutation.seed = 2
#    )

## ---- eval = FALSE------------------------------------------------------------
#  data("fit", package = "gCrisprTools")
#  data("resultsDF", package = "gCrisprTools")
#  
#  fit <- fit[(row.names(fit) %in% row.names(ann)),]
#  resultsDF <- resultsDF[(row.names(resultsDF) %in% row.names(ann)),]

## ---- eval = FALSE------------------------------------------------------------
#  ct.alignmentChart(aln, sk)
#  ct.rawCountDensities(es, sk)

## ---- eval = FALSE------------------------------------------------------------
#  ct.gRNARankByReplicate(es, sk)
#  ct.gRNARankByReplicate(es, sk, annotation = ann, geneSymb = "NoTarget")  #Show locations of NTC gRNAs

## ---- eval = FALSE------------------------------------------------------------
#  ct.viewControls(es, ann, sk, normalize = FALSE)
#  ct.viewControls(es, ann, sk, normalize = TRUE)

## ---- eval = FALSE------------------------------------------------------------
#  ct.GCbias(es, ann, sk)
#  ct.GCbias(fit, ann, sk)

## ---- eval = FALSE------------------------------------------------------------
#  ct.stackGuides(es,
#                 sk,
#                 plotType = "gRNA",
#                 annotation = ann,
#                 nguides = 40)

## ---- eval = FALSE------------------------------------------------------------
#  ct.stackGuides(es,
#                 sk,
#                 plotType = "Target",
#                 annotation = ann)

## ---- eval = FALSE------------------------------------------------------------
#  ct.stackGuides(es,
#                 sk,
#                 plotType = "Target",
#                 annotation = ann,
#                 subset = names(sk)[grep('Expansion', sk)])

## ---- eval = FALSE------------------------------------------------------------
#  ct.guideCDF(es, sk, plotType = "gRNA")
#  ct.guideCDF(es, sk, plotType = "Target", annotation = ann)

## ---- eval = FALSE------------------------------------------------------------
#  ct.topTargets(fit,
#                resultsDF,
#                ann,
#                targets = 10,
#                enrich = TRUE)
#  ct.topTargets(fit,
#                resultsDF,
#                ann,
#                targets = 10,
#                enrich = FALSE)

## ---- eval = FALSE------------------------------------------------------------
#  ct.viewGuides("Target1633", fit, ann)
#  ct.gRNARankByReplicate(es, sk, annotation = ann, geneSymb = "Target1633")

## ---- eval = FALSE------------------------------------------------------------
#  enrichmentResults <-
#    ct.PantherPathwayEnrichment(
#      resultsDF,
#      pvalue.cutoff = 0.01,
#      enrich = TRUE,
#      organism = 'mouse'
#    )

## ---- eval = FALSE------------------------------------------------------------
#  data("essential.genes", package = "gCrisprTools")
#  ROCs <- ct.ROC(resultsDF, essential.genes, stat = "deplete.p")
#  PRCs <- ct.PRC(resultsDF, essential.genes, stat = "deplete.p")

## ---- eval = FALSE------------------------------------------------------------
#  path2report <-      #Make a report of the whole experiment
#    ct.makeReport(fit = fit,
#                  eset = es,
#                  sampleKey = sk,
#                  annotation = ann,
#                  results = resultsDF,
#                  aln = aln,
#                  outdir = ".")
#  
#  path2QC <-          #Or one focusing only on experiment QC
#    ct.makeQCReport(es,
#                    trim = 1000,
#                    log2.ratio = 0.05,
#                    sampleKey = sk,
#                    annotation = ann,
#                    aln = aln,
#                    identifier = 'Crispr_QC_Report',
#                    lib.size = NULL
#                    )
#  
#  path2Contrast <-    #Or Contrast-specific one
#    ct.makeContrastReport(eset = es,
#                          fit = fit,
#                          sampleKey = sk,
#                          results = resultsDF,
#                          annotation = ann,
#                          comparison.id = NULL,
#                          identifier = 'Crispr_Contrast_Report')