Advanced Screen Analysis: Contrast Comparisons
This is a companion vignette briefly describing the advanced capabilites available for comparing, summarizing, and integrating screening contrasts with gCrisprTools, introduced with version 2.0.
Simplified Results Objects
Historically, gCrisprTools was focused on making results data.frames, which exhaustively summarize the results of a screen contrast. They look like this:
## Loading required package: BiocGenerics##
## Attaching package: 'BiocGenerics'## The following objects are masked from 'package:stats':
##
## IQR, mad, sd, var, xtabs## The following objects are masked from 'package:base':
##
## Filter, Find, Map, Position, Reduce, anyDuplicated, aperm, append,
## as.data.frame, basename, cbind, colnames, dirname, do.call,
## duplicated, eval, evalq, get, grep, grepl, intersect, is.unsorted,
## lapply, mapply, match, mget, order, paste, pmax, pmax.int, pmin,
## pmin.int, rank, rbind, rownames, sapply, saveRDS, setdiff, table,
## tapply, union, unique, unsplit, which.max, which.min## Welcome to Bioconductor
##
## Vignettes contain introductory material; view with
## 'browseVignettes()'. To cite Bioconductor, see
## 'citation("Biobase")', and for packages 'citation("pkgname")'.library(gCrisprTools)
knitr::opts_chunk$set(message = FALSE, fig.width = 8, fig.height = 8)
data(resultsDF)
head(resultsDF)## ID target geneID geneSymbol
## Target1106_a Target1106_a TCAGCCGCTCCTACGGATT 1236 Target1106
## Target1106_c Target1106_c GTGCGCTAAGTCGGCAGAT 1236 Target1106
## Target1106_g Target1106_g TAAACTCTGTCGGGTAGAC 1236 Target1106
## Target1106_e Target1106_e TGGTGATTTCTAGGAAGTT 1236 Target1106
## Target1106_b Target1106_b AGACAAGAGCTATCCTATG 1236 Target1106
## Target1106_d Target1106_d CAACTTCGCCTGTTATCCG 1236 Target1106
## gRNA Log2 Fold Change gRNA Depletion P gRNA Depletion Q
## Target1106_a 7.572398 0.99999 1
## Target1106_c 7.589259 1.00000 1
## Target1106_g 7.642459 1.00000 1
## Target1106_e 7.768537 1.00000 1
## Target1106_b 7.569902 1.00000 1
## Target1106_d 7.654991 1.00000 1
## gRNA Enrichment P gRNA Enrichment Q Target-level Enrichment P
## Target1106_a 6.4548e-06 0.0058481 0
## Target1106_c 2.4731e-06 0.0032638 0
## Target1106_g 3.9303e-06 0.0042285 0
## Target1106_e 2.1136e-06 0.0032638 0
## Target1106_b 2.1912e-06 0.0032638 0
## Target1106_d 2.9287e-06 0.0033610 0
## Target-level Enrichment Q Target-level Depletion P
## Target1106_a 0 1
## Target1106_c 0 1
## Target1106_g 0 1
## Target1106_e 0 1
## Target1106_b 0 1
## Target1106_d 0 1
## Target-level Depletion Q Median log2 Fold Change Rho_enrich
## Target1106_a 1 7.580828 9.047675e-25
## Target1106_c 1 7.580828 9.047675e-25
## Target1106_g 1 7.580828 9.047675e-25
## Target1106_e 1 7.580828 9.047675e-25
## Target1106_b 1 7.580828 9.047675e-25
## Target1106_d 1 7.580828 9.047675e-25
## Rho_deplete
## Target1106_a 1
## Target1106_c 1
## Target1106_g 1
## Target1106_e 1
## Target1106_b 1
## Target1106_d 1In many applications, there are limitations to these objects. For example:
- They are reagent-focused, but many downstream applications are focused on Targets
- The
colnamesare human-readable instead of machine readable - The distinction between
geneIDandgeneSymbolis not clear - Many columns are not informative for the majority of modern applications/screens
- Historically, they are sorted to put the strongest signals at the top (this is not the case now)
To help with downstream analyses, we introduce the
simpleResult object
## geneID geneSymbol Rho_enrich Rho_deplete best.p best.q direction
## Target1106 1236 Target1106 9.047675e-25 1 0 0 enrich
## Target1633 1369 Target1633 7.117493e-24 1 0 0 enrich
## Target1631 51316 Target1631 9.377686e-21 1 0 0 enrich
## Target1352 5526 Target1352 7.906212e-19 1 0 0 enrich
## Target1469 639 Target1469 4.826451e-16 1 0 0 enrich
## Target1256 1050 Target1256 3.101526e-11 1 0 0 enrichThese objects are simpler representations of target signals that are applicable for “most” situations (e.g., when the gRNAs associated with a target have similar effects). They are:
- Target focused, but target signals are collapsed to single directional P/Q stats
- Collapsed on a user-specified identifier to enable comparisons across screening technologies
- Both human-readable and machine-readable
- Consistently ordered, and may be easily placed in register with one another
This format enables better comparisons between screens, and we have some helper functions to support this:
# Make "another" result
res2 <- res
res2$best.p <- res2$best.p * runif(nrow(res2))
res2$direction[sample(1:nrow(res), 500)] <- res2$direction[sample(1:nrow(res), 500)]
regularized <- ct.regularizeContrasts(dflist = list('Experiment1' = res[1:1500,],
'Experiment2' = res2[1:1900,]),
collapse = 'geneSymbol')
str(regularized)## List of 2
## $ Experiment1:'data.frame': 1500 obs. of 7 variables:
## ..$ geneID : chr [1:1500] "10059" "2982" "1294" "5641" ...
## ..$ geneSymbol : chr [1:1500] "NoTarget" "Target10" "Target100" "Target1000" ...
## ..$ Rho_enrich : num [1:1500] 0.81 1 1 1 0.238 ...
## ..$ Rho_deplete: num [1:1500] 0.000367 0.097341 0.014417 0.000897 0.227184 ...
## ..$ best.p : num [1:1500] 0.008 0.312 0.078 0.004 0.162 0.403 0.162 0.022 0.133 0.005 ...
## ..$ best.q : num [1:1500] 0.227 0.965 0.694 0.147 1 ...
## ..$ direction : chr [1:1500] "deplete" "deplete" "deplete" "deplete" ...
## $ Experiment2:'data.frame': 1500 obs. of 7 variables:
## ..$ geneID : chr [1:1500] "10059" "2982" "1294" "5641" ...
## ..$ geneSymbol : chr [1:1500] "NoTarget" "Target10" "Target100" "Target1000" ...
## ..$ Rho_enrich : num [1:1500] 0.81 1 1 1 0.238 ...
## ..$ Rho_deplete: num [1:1500] 0.000367 0.097341 0.014417 0.000897 0.227184 ...
## ..$ best.p : num [1:1500] 0.00672 0.1444 0.07347 0.00206 0.08163 ...
## ..$ best.q : num [1:1500] 0.227 0.965 0.694 0.147 1 ...
## ..$ direction : chr [1:1500] "deplete" "deplete" "deplete" "deplete" ...The above function takes in a (named) list of results objects and
creates a list of simpleResults with the same names that
are “in register” with one another, using the shared elements specified
by collapse.
The main utility of this function is twofold. First, it enables comparison of lightweight results objects that are derived from screens that could have been executed with different libraries, systems, or technologies by focusing on the targets and their directional significance measures. Second, this “named list of standardized results” can become a standard structure for encoding grouped contrasts for comparison purposes, allowing us to do more sophisticated comparisons between and across contrasts.
Testing Overlaps Between Two or More Screens
The standardized format allows straightforward identification of
signal overlaps observed within multiple screens via
ct.compareContrasts(). By default, these comparisons are
done conditionally, but users may specify more rigid criteria.
comparison <- ct.compareContrasts(dflist = regularized,
statistics = c('best.q', 'best.p'),
cutoffs = c(0.5,0.05),
same.dir = rep(TRUE, length(regularized)))
head(comparison, 30)## geneID geneSymbol Rho_enrich Rho_deplete best.p best.q
## NoTarget 10059 NoTarget 0.810198260 0.0003666943 0.008 0.2269867
## Target10 2982 Target10 1.000000000 0.0973406417 0.312 0.9651262
## Target100 1294 Target100 1.000000000 0.0144169517 0.078 0.6944937
## Target1000 5641 Target1000 1.000000000 0.0008970760 0.004 0.1467586
## Target1001 323 Target1001 0.238351536 0.2271836123 0.162 1.0000000
## Target1003 11274 Target1003 1.000000000 0.1122383453 0.403 1.0000000
## Target1004 3697 Target1004 0.367507856 0.9733795039 0.162 1.0000000
## Target1005 445 Target1005 1.000000000 0.0067735320 0.022 0.4106667
## Target1006 10874 Target1006 1.000000000 0.0290253030 0.133 0.7877429
## Target1007 2488 Target1007 0.001687259 0.9734058256 0.005 0.3432258
## Target1008 3269 Target1008 0.341020764 0.9926446575 0.223 1.0000000
## Target1009 23114 Target1009 1.000000000 0.0524040002 0.206 0.9113680
## Target101 7398 Target101 0.026185087 0.8184220674 0.043 1.0000000
## Target1014 4057 Target1014 1.000000000 0.2241604775 0.582 1.0000000
## Target1016 2326 Target1016 1.000000000 0.1931948401 0.526 1.0000000
## Target1017 83461 Target1017 0.169090848 0.0428962671 0.115 1.0000000
## Target1019 650 Target1019 1.000000000 0.0353408339 0.150 0.7993234
## Target102 347734 Target102 1.000000000 0.1445274167 0.442 1.0000000
## Target1020 2324 Target1020 1.000000000 0.2345659583 0.574 1.0000000
## Target1021 821 Target1021 0.048472096 0.0031305804 0.022 0.4106667
## Target1022 6533 Target1022 1.000000000 0.2222939519 0.536 1.0000000
## Target1023 6950 Target1023 1.000000000 0.2252680878 0.578 1.0000000
## Target1024 633 Target1024 1.000000000 0.1437704685 0.432 1.0000000
## Target1025 5073 Target1025 0.167112788 0.0901525902 0.134 1.0000000
## Target1026 54468 Target1026 1.000000000 0.2690389752 0.632 1.0000000
## Target1027 9344 Target1027 1.000000000 0.0666379350 0.254 0.9521783
## Target1028 1439 Target1028 0.078493433 0.6994925536 0.073 1.0000000
## Target1029 4481 Target1029 0.007853608 0.9798213353 0.013 0.7476757
## Target103 5167 Target103 1.000000000 0.0379885178 0.167 0.8401324
## Target1030 29106 Target1030 0.089682636 0.2064889045 0.089 1.0000000
## direction replicated
## NoTarget deplete TRUE
## Target10 deplete FALSE
## Target100 deplete FALSE
## Target1000 deplete TRUE
## Target1001 enrich FALSE
## Target1003 deplete FALSE
## Target1004 enrich FALSE
## Target1005 deplete TRUE
## Target1006 deplete FALSE
## Target1007 enrich FALSE
## Target1008 enrich FALSE
## Target1009 deplete FALSE
## Target101 enrich FALSE
## Target1014 deplete FALSE
## Target1016 deplete FALSE
## Target1017 enrich FALSE
## Target1019 deplete FALSE
## Target102 deplete FALSE
## Target1020 deplete FALSE
## Target1021 deplete TRUE
## Target1022 deplete FALSE
## Target1023 deplete FALSE
## Target1024 deplete FALSE
## Target1025 enrich FALSE
## Target1026 deplete FALSE
## Target1027 deplete FALSE
## Target1028 enrich FALSE
## Target1029 enrich FALSE
## Target103 deplete FALSE
## Target1030 enrich FALSEThis function returns a simplifiedResult version of the
mainresult argument, appended with a logical column
indicating whether signals in the mainresult contrast
passing the first significance cutoff are replicated in the
validationresult contrast at the second significance
cutoff. This sort of conditional scoring was shown previously to be
useful in interpreting the results of validation and counterscreens.
For convenience, we can also return summary statistics characterizing the overlap between the screens:
ct.compareContrasts(dflist = regularized,
statistics = c('best.q', 'best.p'),
cutoffs = c(0.5,0.05),
same.dir = rep(TRUE, length(regularized)),
return.stats = TRUE)## expected observed p
## enrich 3.7145 21 0
## deplete 42.6975 130 0
## all 46.4120 151 0This can be useful when exploring appropriate cutoff thresholds, or when asking broader questions about overall congruence.
Visualization of Screen Enrichment and Depletion Dynamics
We provide a number of methods for visualizing and contextualizing the overall signals present within sets of screens. One of the simplest is the cointrast barcharts, which represent the number of observed enrichment and depletion signals in each screen according to specified significance criteria:
In the above figure, each contrast is represented as a horizontal bar, and targets enriched and depleted are represented as bars extending to the right and the left of the vertical dotted line, respectively.
We can also compare the signals observed in two screens directly in a scatterplot:
This provides a scatter plot of the indicated statistic, with quadrants defined according to the user-specified cutoff. The number of targets in each of the quadrants is indicated in grey, and quadrants are keyed like this:
1 2 3 4 5 6 7 8 9
A more complete genewise picture can be achieved by examining the returned invisible object, which appends the relevant quadrants to assist in in focusing on particular targets of interest:
## geneID geneSymbol Rho.enrich.Experiment1 Rho.deplete.Experiment1
## NoTarget 10059 NoTarget 0.09140869 3.4356958
## Target10 2982 Target10 0.00000000 1.0117058
## Target100 1294 Target100 0.00000000 1.8411266
## Target1000 5641 Target1000 0.00000000 3.0471708
## Target1001 323 Target1001 0.62278205 0.6436230
## Target1003 11274 Target1003 0.00000000 0.9498587
## p.Experiment1 q.Experiment1 Rho.enrich.Experiment2
## NoTarget 2.0705811 0.594707067 0.09140869
## Target10 0.5051500 0.003309628 0.00000000
## Target100 1.1051303 0.141597698 0.00000000
## Target1000 2.3467875 0.759349249 0.00000000
## Target1001 0.7891466 0.000000000 0.62278205
## Target1003 0.3941565 0.000000000 0.00000000
## Rho.deplete.Experiment2 p.Experiment2 q.Experiment2 quadrant
## NoTarget 3.4356958 2.1714058 0.594707067 7
## Target10 1.0117058 0.8403591 0.003309628 5
## Target100 1.8411266 1.1337325 0.141597698 5
## Target1000 3.0471708 2.6814611 0.759349249 7
## Target1001 0.6436230 1.0880364 0.000000000 5
## Target1003 0.9498587 0.6243094 0.000000000 5
## x y
## NoTarget -2.0705811 -2.1714058
## Target10 -0.5051500 -0.8403591
## Target100 -1.1051303 -1.1337325
## Target1000 -2.3467875 -2.6814611
## Target1001 0.7891466 1.0880364
## Target1003 -0.3941565 -0.6243094As an aside, this simplified infrastructure allows straightforward dissection of interaction effects as well. Often, it is helpful to identify targets that are enriched or depleted with respect to one contrast but inactive with respect to another (e.g., to identify genes that impact a screening phenotype but do not deplete over time).
library(Biobase)
library(limma)
library(gCrisprTools)
#Create a complex model design; removing the replicate term for clarity
data("es", package = "gCrisprTools")
data("ann", package = "gCrisprTools")
design <- model.matrix(~ 0 + TREATMENT_NAME, pData(es))
colnames(design) <- gsub('TREATMENT_NAME', '', colnames(design))
contrasts <-makeContrasts(ControlTime = ControlExpansion - ControlReference,
DeathOverTime = DeathExpansion - ControlReference,
Interaction = DeathExpansion - ControlExpansion,
levels = design)
es <- ct.normalizeGuides(es, method = "scale") #See man page for other options
vm <- voom(exprs(es), design)
fit <- lmFit(vm, design)
fit <- contrasts.fit(fit, contrasts)
fit <- eBayes(fit)
allResults <- sapply(colnames(fit$coefficients),
function(x){
ct.generateResults(fit,
annotation = ann,
RRAalphaCutoff = 0.1,
permutations = 1000,
scoring = "combined",
permutation.seed = 2,
contrast.term = x)
}, simplify = FALSE)
allSimple <- ct.regularizeContrasts(allResults)Using the logical columns we can Identify relevant sets of targets. For example, the number of targets changing over time in both timecourses:
time.effect <- ct.compareContrasts(list("con" = allSimple$ControlTime,
"tx" = allSimple$DeathOverTime))
summary(time.effect$replicated)## Mode FALSE TRUE
## logical 2012 116or targets with a toxicity modifying effect that compromise intrinsic viability:
mod.control <- ct.compareContrasts(list("con" = allSimple$ControlTime,
"Interaction" = allSimple$Interaction),
same.dir = c(TRUE, FALSE))
summary(mod.control$replicated)## Mode FALSE TRUE
## logical 2075 53More Comparisons
Sometimes you might be curious about the relationship between many contrasts. You can accomplish this by making an UpSet plot:
In addition to constructing the UpSet plot above, by default we include fold enrichment and p-value estimates to help interpret the various bars in the context of the nominal or conditional expected values.
Finally, the above function returns a combination matrix containing the overlap values and associated targets, which can be useful for interrogating intersection sets of interest.
## A combination matrix with 3 sets and 7 combinations.
## ranges of combination set size: c(21, 165).
## mode for the combination size: conditional.
## sets are on rows.
##
## Combination sets are:
## ControlTime DeathOverTime Interaction code size
## x x x 111 28
## x x 110 116
## x x 101 21
## x x 011 70
## x 100 165
## x 010 110
## x 001 55
##
## Sets are:
## set size
## ControlTime 2128
## DeathOverTime 2128
## Interaction 2128See the documentation about combination matrices provided in the
ComplexHeatmap package for accessor functions and
additional information about the structure and use of this object.
Ontological Enrichment
Though not subject to specific set-level analyses yet, the
ct.seas() function can be likewise extended to use lists of
results. The standardized objects can then be consolidated to ask
broader questions about enrichment and depletion using standard
methods:
genesetdb <- sparrow::getMSigGeneSetDb(collection = 'h', species = 'human', id.type = 'entrez')
sparrowList <- ct.seas(allSimple, gdb = genesetdb)## Warning in ora(logFC, gsd, selected = selected, groups = groups, feature.bias =
## feature.bias, : BiasedUrn package not installed, running in unbiased mode ...## Warning in preparePathwaysAndStats(pathways, stats, minSize, maxSize, gseaParam, : There are ties in the preranked stats (35.12% of the list).
## The order of those tied genes will be arbitrary, which may produce unexpected results.## Warning in ora(logFC, gsd, selected = selected, groups = groups, feature.bias =
## feature.bias, : BiasedUrn package not installed, running in unbiased mode ...## Warning in preparePathwaysAndStats(pathways, stats, minSize, maxSize, gseaParam, : There are ties in the preranked stats (65.07% of the list).
## The order of those tied genes will be arbitrary, which may produce unexpected results.## Warning in ora(logFC, gsd, selected = selected, groups = groups, feature.bias =
## feature.bias, : BiasedUrn package not installed, running in unbiased mode ...## Warning in preparePathwaysAndStats(pathways, stats, minSize, maxSize, gseaParam, : There are ties in the preranked stats (63.94% of the list).
## The order of those tied genes will be arbitrary, which may produce unexpected results.## $ControlTime
## SparrowResult (max FDR by collection set to 0.20%)
## ---------------------------------------------------
## collection method geneset_count sig_count sig_up sig_down
## 1 H ora 50 0 0 0
## 2 H ora.deplete 50 0 0 0
## 3 H ora.enrich 50 0 0 0
## 4 H fgsea 50 0 0 0
##
##
## $DeathOverTime
## SparrowResult (max FDR by collection set to 0.20%)
## ---------------------------------------------------
## collection method geneset_count sig_count sig_up sig_down
## 1 H ora 50 0 0 0
## 2 H ora.deplete 50 0 0 0
## 3 H ora.enrich 50 0 0 0
## 4 H fgsea 50 1 0 1
##
##
## $Interaction
## SparrowResult (max FDR by collection set to 0.20%)
## ---------------------------------------------------
## collection method geneset_count sig_count sig_up sig_down
## 1 H ora 50 0 0 0
## 2 H ora.deplete 50 1 0 1
## 3 H ora.enrich 50 0 0 0
## 4 H fgsea 50 1 0 1#Can use returned matrices to facilitate downstream comparisons:
plot(-log10(sparrow::results(sparrowList$DeathOverTime, 'fgsea')$padj),
-log10(sparrow::results(sparrowList$Interaction, 'fgsea')$padj),
pch = 19, col = rgb(0,0,0.8,0.5),
ylab = "Pathway -log10(P), Treatment Over Time",
xlab = "Pathway -log10(P), Marginal Time Effect, Treatment Arm",
main = 'Evidence for Pathway Enrichment')
abline(0,1,lty = 2)
## R version 4.4.1 (2024-06-14)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.1 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
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## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
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## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
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## time zone: Etc/UTC
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] limma_3.61.12 gCrisprTools_2.13.0 Biobase_2.67.0
## [4] BiocGenerics_0.53.0
##
## loaded via a namespace (and not attached):
## [1] DBI_1.2.3 gridExtra_2.3
## [3] GSEABase_1.67.1 BiocSet_1.21.0
## [5] rlang_1.1.4 magrittr_2.0.3
## [7] clue_0.3-65 GetoptLong_1.0.5
## [9] msigdbr_7.5.1 sparrow_1.11.0
## [11] matrixStats_1.4.1 compiler_4.4.1
## [13] RSQLite_2.3.7 png_0.1-8
## [15] vctrs_0.6.5 pkgconfig_2.0.3
## [17] shape_1.4.6.1 crayon_1.5.3
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