Example Workflow For Processing a Single Pooled Screen

Example Workflow For Processing a Single Screen

This is an example workflow for processing a pooled screening eperiment using the provided sample data. See the various manpages for additional visualization options and algorithmic details.

Note that what follows describes a very basic analysis. If you are considering integrating the results of many different screen contrasts or even different experiments and/or technologies, refer to the Advanced Screen Analysis: Contrast Comparisons vignette.

Load dependencies and data

suppressPackageStartupMessages(library(Biobase))
suppressPackageStartupMessages(library(limma))
suppressPackageStartupMessages(library(gCrisprTools))

data("es", package = "gCrisprTools")
data("ann", package = "gCrisprTools")
data("aln", package = "gCrisprTools")
knitr::opts_chunk$set(message = FALSE, fig.width = 8, fig.height = 8, warning = FALSE)

Make a sample key, structured as a factor with control samples in the first level

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

Generate a contrast of interest using voom/limma; pairing replicates is a good idea if that information is available.

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

Optionally, trim of trace reads from the unnormalized object (see man page for details)

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

Normalize, convert to a voom object, and generate a contrast

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)

Edit the annotation file if you used ct.filterReads above

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

Summarize gRNA signals to identify target genes of interest

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

Alternative Annotations

In some cases, reagents might target multiple known elements (e.g., gRNAs in a CRISPRi library that target multiple promoters of the same gene). In such cases, you can specify this via the alt.annotation argument to ct.generateResults(). Alternative annotations are supplied as a list of character vectors named for the reagents.

# Create random alternative target associations 

altann <- sapply(ann$ID, 
                 function(x){
                   out <- as.character(ann$geneSymbol)[ann$ID %in% x]
                   if(runif(1) < 0.01){out <- c(out, sample(as.character(ann$geneSymbol), size = 1))}
                   return(out)
                 }, simplify = FALSE)

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

Optionally, just load an example results object for testing purposes (trimming out reads as necessary)

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)),]

targetResultDF <- ct.simpleResult(resultsDF) #For a simpler target-level result object

Quality Control

gCrisprTools contains a variety of pooled screen-specific quality control and visualization tools (see man pages for details):

ct.alignmentChart(aln, sk)

ct.rawCountDensities(es, sk)

Visualize gRNA abundance distributions

ct.gRNARankByReplicate(es, sk) 

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

Visualize control guide behavior across conditions

ct.viewControls(es, ann, sk, normalize = FALSE)

ct.viewControls(es, ann, sk, normalize = TRUE)

Visualize GC bias across samples, or within an experimental contrast

ct.GCbias(es, ann, sk)

ct.GCbias(fit, ann, sk)

View most variable gRNAs/Genes (as % of sequencing library)

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

ct.stackGuides(es, 
               sk, 
               plotType = "Target", 
               annotation = ann)

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

View a CDF of genes/guides

ct.guideCDF(es, sk, plotType = "gRNA")

ct.guideCDF(es, sk, plotType = "Target", annotation = ann)

Target-Level Visualization and Analysis

View the overall enrichment and depletion trends identified in the screen:

ct.contrastBarchart(resultsDF)

View top enriched/depleted candidates

ct.topTargets(fit,
              resultsDF,
              ann,
              targets = 10,
              enrich = TRUE)

ct.topTargets(fit,
              resultsDF,
              ann,
              targets = 10,
              enrich = FALSE)

View the behavior of reagents targeting a particular gene of interest

ct.viewGuides("Target1633", fit, ann)

ct.gRNARankByReplicate(es, sk, annotation = ann, geneSymb = "Target1633")