Competitive screening experiments, in which bulk cell cultures infected with a heterogeneous viral library are experimentally manipulated to identify guide RNAs or shRNAs that influence cell viability, are conceptually straightforward but often challenging to implement. Here, we present gCrisprTools, an R/Bioconductor analysis suite facilitating quality assessment, target prioritization, and interpretation of arbitrarily complex competitive screening experiments. gCrisprTools provides functionalities for detailed and principled analysis of diverse aspects of these experiments both as a standalone pipeline or as an extension to alternative analytical approaches.
Install gCrisprTools in the usual way:
If you use gCrisprTools while developing a publication, please cite the following paper:
[Bioinformatics App Note Citation, to be updated later]
This vignette is only one of the resources provided in
gCrisprTools to help you understand, analyse, and explore pooled screening data. As appropriate, please see the
/vignettes subdirectory for additional documentation describing example code, and the
/inst directory for more information about algorithm implementation and package layout.
gCrisprTools uses the existing
Biobase framework for data storage and manipulation and consequently depends heavily on the
library(Biobase) library(limma) library(gCrisprTools)
To use the various methods available in this package, you will first need to conform your screen data into an
ExpressionSet object containing cassette abundance counts in the assayData slot, retrievable with
exprs(). This package assumes that end users are familiar enough with the R/Bioconductor framework and their own sequencing pipelines to extract raw cassette counts from FASTQ files and to compose them into an
ExpressionSet. For newer users read counting may be facilitated with cutadapt or other software designed for these purposes; details about composition of
ExpressionSet objects can be found in the Biobase vignette.
Raw cassette counts should be contained within an
ExpressionSet object, with the counts retrievable with
exprs(). The column names (
colnames()) should correspond to unique sample identifiers, and the row names (
row.names()) should correspond to identifiers uniquely specifying each cassette of interest.
data("es", package = "gCrisprTools") es head(exprs(es))
gCrisprTools requires an annotation object mapping the individual cassettes to genes or other genomic features for most applications. The annotation object should be provided as a named
data.frame, with columns describing the '
geneID' and '
geneSymbol' of the target elements to which each cassette is annotated. These columns should contain character vectors with elements that uniquely describe the targets in the screen; by convention, the
geneID field contains an official identifier that unambiguously describes each target element in a manner suitable for external software (e.g., an Entrez ID). The
geneSymbol column indicates a more human-readable descriptor, such as a gene symbol.
The annotation object may optionally contain other columns with additional information about the corresponding cassettes.
data("ann", package = "gCrisprTools") head(ann)
gCrisprTools functions require or are enhanced by a sample key detailing the experimental groups of the functions included in the study. This key should be provided as a named factor, with
names perfectly matching the
colnames of the ExpressionSet. The first level of the sample key should correspond to the 'control' condition, indexing samples whose cassette distributions are expected to be the minimally distorted by experimental treatments.
sk <- relevel(as.factor(pData(es)$TREATMENT_NAME), "ControlReference") names(sk) <- row.names(pData(es)) sk
Users may provide a matrix of alignment statistics to enhance some applications, including QC reporting. These should be provided as a numeric matrix in which rows correspond to
targets (reads containing a target cassette),
nomatch (reads containing a cassette sequence but not a known target sequence),
rejections (reads not containg a cassette sequence), and
double_match (reads derived from multiple cassettes). The column names should exactly match the
colnames() of the ExpressionSet object. Simple charting functionality is also provided to inspect the alignment rates of each sample.
data("aln", package = "gCrisprTools") head(aln) ct.alignmentChart(aln, sk)
gCrisprTools provides tools for common data preprocessing steps, including eliminating underinfected or contaminant cassettes and sample-level normalization.
Low abundance cassettes can be removed by specifying a minimum number of counts or a level relative to the trimmed distribution maximum.
es.floor <- ct.filterReads(es, read.floor = 30, sampleKey = sk) es <- ct.filterReads(es, trim = 1000, log2.ratio = 4, sampleKey = sk) ##Convenience function for conforming the annotation object to exclude the trimmed gRNAs ann <- ct.prepareAnnotation(ann, es, controls = "NoTarget")
A suite of normalization tools are provided with the
ct.normalizeGuides() wrapper function; see the relevant manual pages for further details about these methods.
es <- ct.normalizeGuides(es, 'scale', annotation = ann, sampleKey = sk, plot.it = TRUE) es.norm <- ct.normalizeGuides(es, 'slope', annotation = ann, sampleKey = sk, plot.it = TRUE) es.norm <- ct.normalizeGuides(es, 'controlScale', annotation = ann, sampleKey = sk, plot.it = TRUE, geneSymb = 'NoTarget') es.norm <- ct.normalizeGuides(es, 'controlSpline', annotation = ann, sampleKey = sk, plot.it = TRUE, geneSymb = 'NoTarget')
For convenience, experiment-level dynamics and the effects of various preprocessing steps may be automatically summarized in the form of a report. The following code isn't run as part of this vignette, but if run from the terminal
path2QC will indicate the path to an html Quality Control report.
#Not run: path2QC <- ct.makeQCReport(es, trim = 1000, log2.ratio = 0.05, sampleKey = sk, annotation = ann, aln = aln, identifier = 'Crispr_QC_Report', lib.size = NULL )
gCrisprTools package provides a series of functions for assessing distributional and technical properties of sequencing libraries. Please see additional details about all of these methods on their respective manual pages.
The raw cassette count distributions can be visualized to determine whether samples were inadequately sequenced or if PCR amplification artifacts might be present.
Aspects of cassette distributions are often better visualized with a 'waterfall' plot than a standard density estimate, which can clarify the ranking relationships in specific parts of the distribution.
ct.gRNARankByReplicate(es, sk) #Visualization of gRNA abundance distribution
These plots also enable explicit visualization of cassettes of interest in the context of the experimental distribution.
ct.gRNARankByReplicate(es, sk, annotation = ann, geneSymb = "Target1633")
gCrisprTools provides tools for visualizing the behavior of control gRNAs across experimental conditions.
ct.viewControls(es, ann, sk, normalize = FALSE, geneSymb = 'NoTarget')
Depending on the screen in question, it can be useful to quantify the extent to which experimental libraries have been distorted by experimental treatments.
gCrisprTools provides tools to estimate an empirical cumulative distribution function describing the cassettes or (targets) within a screen.
ct.guideCDF(es, sk, plotType = "gRNA")
The core analytical machinery of gCrisprTools is built on the linear modelling framework implemented in the
limma package. Specifically, users employ
limma/voom to generate an experimental contrast of interest at the gRNA level. The model coefficent and P-value estimates may be subsequently processed with the infrastructure provided by
design <- model.matrix(~ 0 + REPLICATE_POOL + TREATMENT_NAME, pData(es)) colnames(design) <- gsub('TREATMENT_NAME', '', colnames(design)) contrasts <-makeContrasts(DeathExpansion - ControlExpansion, levels = design) vm <- voom(exprs(es), design) fit <- lmFit(vm, design) fit <- contrasts.fit(fit, contrasts) fit <- eBayes(fit)
After generating a fit object (class
MArrayLM) for a contrast of interest, we may summarize the signals from the various cassettes annotated to each target via RRA$\alpha$ aggregation. The core algorithm is described in detail in the original publication on Robust Rank Aggregation[^1] and has been implemented according to the $\alpha$ thresholding modification proposed by Li et al.[^2] Briefly, gRNA signals contained in the specified fit object are ranked and normalized, and these ranks are grouped by the associated target and assigned a score ($\rho$) on the basis of the skewness of the gRNA signal ranks. The statistical significance of each target-level score is then assessed by permutation of the gRNA target assignments. Q-values are computed directly from the resulting P-value distributions using the FDR approach described by Benjamini and Hochberg.[^3]
A more extensive treatment of RRA$\alpha$ and comparisons to MAGeCK may be found in
resultsDF <- ct.generateResults( fit, annotation = ann, RRAalphaCutoff = 0.1, permutations = 1000, scoring = "combined" )
The resulting dataframe contains columns passing some of the information from the fit and annotation objects, as well as a number of statistics describing the evidence for a target's depletion or enrichment within the context of the screen. These include the Target-level P and Q values quantifying the evidence for enrichment or depletion, the median log2 fold change of all of the gRNAs associated with each target, and the rankings of the target-level $/rho$ statistics (gene-level scores may be useful for ranking targets with equivalent P-values).
After identifying candidate targets, various aspects of the contrast may be visualized with
ct.topTargets function enables simple visualization of the model effect estimates (log2 fold changes) and associated uncertainties of all cassettes associated with the top-ranking targets.
ct.topTargets(fit, resultsDF, ann, targets = 10, enrich = TRUE)
In some screens it can be useful to visualize the degree of library distortion associated with the strongest signals. Such an approach can supply additional confidence in a particular candidate of interest by showing that clear differences are evident outside of the linear modeling framework (which may be inaccurate in heavily distorted libraries).
ct.stackGuides( es, sk, plotType = "Target", annotation = ann, subset = names(sk)[grep('Expansion', sk)] )
gCrisprTools provides methods to visualize the behavior of individual cassettes annotated to target of interest, and positions these within the observed distribution of effect sizes across all cassettes within the experiment.
ct.viewGuides("Target1633", fit, ann)
As with the Quality Control components of an individual screen,
gCrisprTools provides functionality to automatically generate contrast-level reports.
#Not run: path2Contrast <- ct.makeContrastReport(eset = es, fit = fit, sampleKey = sk, results = resultsDF, annotation = ann, comparison.id = NULL, identifier = 'Crispr_Contrast_Report')
If you wish, you can also make a single report encompassing both quality control and the contrast of interest.
#Not run: path2report <- ct.makeReport(fit = fit, eset = es, sampleKey = sk, annotation = ann, results = resultsDF, aln = aln, outdir = ".")
In addition to identifying targets of interest within a screen, it may be worthwhile to ask more comprehensive questions about the targets identified.
gCrisprTools provides a series of basic functions for determining the enrichment of known or unknown target groups within the context of a screen.
If a screen was performed with a library targeting genes,
gCrisprTools can provide basic ontological enrichment testing. This function annotates Entrez gene IDs contained in the
geneID column of the annotation object to pathways contained in the PANTHER database, and then checks for significant enrichment or depletion of these pathways using a hypergeometric test.
#Not run: enrichmentResults <- ct.PantherPathwayEnrichment( resultsDF, pvalue.cutoff = 0.01, enrich = TRUE, organism = 'mouse' ) > head(enrichmentResults) #Note: Pathway names have been edited for display purposes. PATHWAY nGenes sigGenes expected odds p FDR 1 EGF receptor signaling pathway 200 14 5.240550 3.332647 0.0004498023 0.03958260 2 FGF signaling pathway 230 14 5.949958 2.869779 0.0016284304 0.07165094 3 Insulin/MAP kinase cascade 138 9 3.714916 2.785331 0.0101632822 0.20272465 4 CCKR signaling map ST 331 15 8.211061 2.148459 0.0126368744 0.20272465 5 p38 MAPK pathway 145 9 3.891333 2.641968 0.0135928688 0.20272465 6 B cell activation 204 11 5.336192 2.368705 0.0146442541 0.20272465
In some cases, it may be useful to ask whether a set of known targets is disproportionately enriched or depleted within a screen.
gCrisprTools provides functions for answering these sorts of questions with
ct.ROC(), which generates Reciever-Operator Characteristics for a specified gene set within a screen, and
ct.PRC(), which draws precision-recall curves. When called, both functions return the raw data necessary to reproduce or combine these results, along with appropriate statistics for assessing the significance of the overall signal within the specified target set (via a hypergeometric test).
data("essential.genes", package = "gCrisprTools") #Artificial list created for demonstration data("resultsDF", package = "gCrisprTools") ROC <- ct.ROC(resultsDF, essential.genes, stat = "deplete.p") str(ROC)
PRC <- ct.PRC(resultsDF, essential.genes, stat = "deplete.p") str(PRC)
Alternatively, the significance of the enrichment within the target set may be assessed directly with
targetsTest <- ct.targetSetEnrichment(resultsDF, essential.genes, enrich = FALSE) str(targetsTest)
[^1]: Kolde R, Laur S, Adler P, Vilo J. Robust rank aggregation for gene list integration and meta-analysis. Bioinformatics. 2012;28(4):573-80. PMID:22247279
[^2]: Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, Irizarry RA, Liu JS, Brown M, Liu XS. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15(12):554. PMID:25476604
[^3]:Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B. 1995;57(1):289–300. MR 1325392.
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