Examples
qvalue object
The qvalue package is a popular package to estimate q-values and local false discovery rates. To get started, we can load the hedenfalk dataset included in the qvalue package:
## [1] "call" "pi0" "qvalues" "pvalues" "lfdr"
## [6] "pi0.lambda" "lambda" "pi0.smooth"qobj is a qvalue object, generated from the p-values contained in the hedenfalk dataset. If we wanted to use a package such as dplyr or ggplot, we would need to convert the results into a data frame. The biobroom package makes this conversion easy by using the tidy, augment and glance functions:
tidyreturns one row for each choice of the tuning parameter lambda.augmentreturns one row for each provided p-value, including the computed q-value and local false discovery rate.glancereturns a single row containing the estimatedpi0.
Applying these functions to qobj:
## # A tibble: 6 x 3
## lambda smoothed pi0
## <dbl> <lgl> <dbl>
## 1 0.05 FALSE 0.852
## 2 0.1 FALSE 0.807
## 3 0.15 FALSE 0.782
## 4 0.2 FALSE 0.756
## 5 0.25 FALSE 0.731
## 6 0.3 FALSE 0.714## # A tibble: 6 x 3
## p.value q.value lfdr
## <dbl> <dbl> <dbl>
## 1 0.0121 0.0882 0.168
## 2 0.0750 0.209 0.413
## 3 0.995 0.668 1
## 4 0.0418 0.162 0.309
## 5 0.846 0.632 1
## 6 0.252 0.372 0.692## # A tibble: 1 x 2
## pi0 lambda
## <dbl> <dbl>
## 1 0.670 0.95The original data, or in this example the gene names, can be inputted into augment using the data argument:
## # A tibble: 6 x 4
## gene p.value q.value lfdr
## <int> <dbl> <dbl> <dbl>
## 1 1 0.0121 0.0882 0.168
## 2 2 0.0750 0.209 0.413
## 3 3 0.995 0.668 1
## 4 4 0.0418 0.162 0.309
## 5 5 0.846 0.632 1
## 6 6 0.252 0.372 0.692The tidied data can be used to easily create plots:
library(ggplot2)
# use augmented data to compare p-values to q-values
ggplot(augment(qobj), aes(p.value, q.value)) + geom_point() +
ggtitle("Simulated P-values versus Computed Q-values") + theme_bw()
Additionally, we can extract out important information such as significant genes under a false discovery rate threshold:
library(dplyr)
# Find significant genes under 0.05 threshold
sig.genes <- augment(qobj) %>% filter(q.value < 0.05)
head(sig.genes)## # A tibble: 6 x 3
## p.value q.value lfdr
## <dbl> <dbl> <dbl>
## 1 0.000713 0.0232 0.0365
## 2 0.00175 0.0365 0.0597
## 3 0.00265 0.0429 0.0751
## 4 0.00116 0.0301 0.0476
## 5 0.00298 0.0448 0.0802
## 6 0.00235 0.0397 0.0703DESeq2 objects
To demonstrate tidying on DESeq2 objects we have used the published airway RNA-Seq experiment, available as a package from Bioconductor:
if (!requireNamespace("BiocManager", quietly=TRUE))
install.packages("BiocManager")
BiocManager::install("airway")Import the airway dataset:
airway_se is a SummarizedExperiment object, which is a type of object used by the DESeq2 package. Next, we create a DESeqDataSet object and show the output of tidying this object:
## # A tibble: 6 x 3
## gene sample count
## <chr> <chr> <int>
## 1 ENSG00000000003 SRR1039508 679
## 2 ENSG00000000005 SRR1039508 0
## 3 ENSG00000000419 SRR1039508 467
## 4 ENSG00000000457 SRR1039508 260
## 5 ENSG00000000460 SRR1039508 60
## 6 ENSG00000000938 SRR1039508 0Only the gene counts are outputted since there has been no analysis performed. We perform an analysis on the data and then tidy the resulting object:
# differential expression analysis
deseq <- DESeq(airway_dds)
results <- results(deseq)
# tidy results
tidy_results <- tidy(results)
head(tidy_results)## # A tibble: 6 x 7
## gene baseMean estimate stderror statistic p.value p.adjusted
## <chr> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl>
## 1 ENSG00000000003 709. 0.381 0.101 3.79 0.000152 0.00128
## 2 ENSG00000000005 0 NA NA NA NA NA
## 3 ENSG00000000419 520. -0.207 0.112 -1.84 0.0653 0.197
## 4 ENSG00000000457 237. -0.0379 0.143 -0.264 0.792 0.911
## 5 ENSG00000000460 57.9 0.0882 0.287 0.307 0.759 0.895
## 6 ENSG00000000938 0.318 1.38 3.50 0.394 0.694 NAAs an example to show how easy it is to manipulate the resulting object, tidy_results, we can use ggplot2 to create a volcano plot of the p-values:
ggplot(tidy_results, aes(x=estimate, y=log(p.value),
color=log(baseMean))) + geom_point(alpha=0.5) +
ggtitle("Volcano Plot For Airway Data via DESeq2") + theme_bw()
##edgeR objects
Here we use the hammer dataset included in biobroom package. edgeR can be used to perform differential expression analysis as follows:
library(edgeR)
data(hammer)
hammer.counts <- Biobase::exprs(hammer)[, 1:4]
hammer.treatment <- Biobase::phenoData(hammer)$protocol[1:4]
y <- DGEList(counts=hammer.counts,group=hammer.treatment)
y <- calcNormFactors(y)
y <- estimateCommonDisp(y)
y <- estimateTagwiseDisp(y)
et <- exactTest(y)The results of the analysis are stored in et, which is an DGEExact object. We can tidy this object using biobroom:
## # A tibble: 6 x 4
## gene estimate logCPM p.value
## <chr> <dbl> <dbl> <dbl>
## 1 ENSRNOG00000000001 2.65 1.49 0.00000131
## 2 ENSRNOG00000000007 -0.409 -0.226 1
## 3 ENSRNOG00000000008 2.22 -0.407 0.129
## 4 ENSRNOG00000000009 0 -1.31 1
## 5 ENSRNOG00000000010 0.0331 1.79 1
## 6 ENSRNOG00000000012 -3.39 0.0794 0.00375glance shows a summary of the experiment: the number of genes found significant (at a specified alpha), and which contrasts were compared to get the results.
## significant comparison
## 1 6341 control/L5 SNLAdditionally, we can can easily manipulate the resulting object and create a volcano plot of the p-values using ggplot2:
ggplot(tidy(et), aes(x=estimate, y=log(p.value), color=logCPM)) +
geom_point(alpha=0.5) + ggtitle("Volcano Plot for Hammer Data via EdgeR") +
theme_bw()
##limma objects
To demonstrate how biobroom works with limma objects, we generate some simulated data to test the tidier for limma objects.
# create random data and design
dat <- matrix(rnorm(1000), ncol=4)
dat[, 1:2] <- dat[, 1:2] + .5 # add an effect
rownames(dat) <- paste0("g", 1:nrow(dat))
des <- data.frame(treatment = c("a", "a", "b", "b"),
confounding = rnorm(4))We then use lmFit and eBayes (functions included in limma) to fit a linear model and use tidy to convert the resulting object into tidy format:
lfit <- lmFit(dat, model.matrix(~ treatment + confounding, des))
eb <- eBayes(lfit)
head(tidy(lfit))## # A tibble: 6 x 3
## gene term estimate
## <chr> <fct> <dbl>
## 1 g1 treatmentb 1.05
## 2 g2 treatmentb -1.20
## 3 g3 treatmentb -1.33
## 4 g4 treatmentb -3.00
## 5 g5 treatmentb 0.647
## 6 g6 treatmentb -0.122## # A tibble: 6 x 6
## gene term estimate statistic p.value lod
## <chr> <fct> <dbl> <dbl> <dbl> <dbl>
## 1 g1 treatmentb 1.05 0.623 0.534 -5.32
## 2 g2 treatmentb -1.20 -0.712 0.477 -5.28
## 3 g3 treatmentb -1.33 -0.788 0.431 -5.23
## 4 g4 treatmentb -3.00 -1.78 0.0759 -4.17
## 5 g5 treatmentb 0.647 0.384 0.701 -5.43
## 6 g6 treatmentb -0.122 -0.0728 0.942 -5.48Analysis can easily be performed from the tidied data. The package ggplot2 can be used to make a volcano plot of the p-values:
ggplot(tidy(eb), aes(x=estimate, y=log(p.value), color=statistic)) +
geom_point() + ggtitle("Nested Volcano Plots for Simulated Data Processed with limma") +
theme_bw()
##ExpressionSet objects
tidy can also be run directly on ExpressionSet objects, as described in another popular Bioconductor package Biobase. The hammer dataset we used above (which is included in the biobroom package) is an ExpressionSet object, so we’ll use that to demonstrate.
## # A tibble: 6 x 3
## gene sample value
## <chr> <chr> <int>
## 1 ENSRNOG00000000001 SRX020102 2
## 2 ENSRNOG00000000007 SRX020102 4
## 3 ENSRNOG00000000008 SRX020102 0
## 4 ENSRNOG00000000009 SRX020102 0
## 5 ENSRNOG00000000010 SRX020102 19
## 6 ENSRNOG00000000012 SRX020102 7We can add the phenotype information by using the argument addPheno:
## # A tibble: 6 x 8
## gene sample sample.id num.tech.reps protocol strain Time value
## <chr> <chr> <fct> <dbl> <fct> <fct> <fct> <int>
## 1 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 2
## 2 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 4
## 3 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 0
## 4 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 0
## 5 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 19
## 6 ENSRNOG0~ SRX020~ SRX020102 1 control Sprague ~ 2 mo~ 7Now we can easily visualize the distribution of counts for each protocol by using ggplot2:
ggplot(tidy(hammer, addPheno=TRUE), aes(x=protocol, y=log(value))) +
geom_boxplot() + ggtitle("Boxplot Showing Effect of Protocol on Expression")
##MSnSet Objects
tidy can also be run directly on MSnSet objects from MSnbase, which as specialised containers for quantitative proteomics data.
## # A tibble: 6 x 3
## protein sample.id value
## <chr> <chr> <dbl>
## 1 X1 iTRAQ4.114 1348.
## 2 X10 iTRAQ4.114 740.
## 3 X11 iTRAQ4.114 27638.
## 4 X12 iTRAQ4.114 31893.
## 5 X13 iTRAQ4.114 26144.
## 6 X14 iTRAQ4.114 6448.## # A tibble: 6 x 5
## protein sample mz reporters value
## <fct> <fct> <dbl> <fct> <dbl>
## 1 X1 iTRAQ4.114 114. iTRAQ4 1348.
## 2 X10 iTRAQ4.114 114. iTRAQ4 740.
## 3 X11 iTRAQ4.114 114. iTRAQ4 27638.
## 4 X12 iTRAQ4.114 114. iTRAQ4 31893.
## 5 X13 iTRAQ4.114 114. iTRAQ4 26144.
## 6 X14 iTRAQ4.114 114. iTRAQ4 6448.