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User Manual: IgGeneUsage
In IgGeneUsage: Differential gene usage in immune repertoires
knitr::opts_chunk$set(comment = FALSE,
warning = FALSE,
message = FALSE)
require(IgGeneUsage)
require(knitr)
require(ggplot2)
require(ggforce)
require(gridExtra)
require(ggrepel)
require(rstan)
require(reshape2)
rstan_options(auto_write = TRUE)
Introduction
Decoding the properties of immune repertoires is key in understanding the
response of adaptive immunity to challenges such as viral infection. One
important property is biases in Ig gene usage between biological conditions
(e.g. healthy vs turmor). Yet, most analyses for differential gene usage are
performed qualitatively, or with inadequate statistical methods. Here we
introduce IgGeneUsage, a computational tool for the analysis of differential
gene usage.
Input
The input to IgGeneUsage is a data.frame object with usage frequencies for each
gene of a repertoire that belongs to a particular biological condition. The
usage data.frame has the following 4 columns:
- sample_id: identifier of the repertoire (e.g. Patient-1)
- condition: identifier of the condition to which each repertoire
belongs (e.g. healthy or tumor)
- gene_name: specific gene name (e.g. IGHV1-10 or family TRVB1)
- gene_usage_count: numeric (count) of usage related to columns 1-3
The sum of all gene usage counts (column 4) for a given repertoire should be
equal to the total gene usage in that repertoire.
Method
IgGeneUsage transforms the provided input in the following way. Given $R$
repertoires, each having $G$ genes, IgGeneUsage generates a gene usage matrix
$Y^{R \times G}$. Row sums in $Y$ define the total usage in each repertoire
($N$). The design variable $X$ is set to $X = 1$ for repertoires that belong to
the first condition, and $X = -1$ otherwise.
For the analysis of DGU between two biological conditions, we designed the
following Bayesian model ($M$) for zero-inflated beta-binomial regression. This
model can fit over-dispersed gene usage data. The immune repertoire data is also
not exhaustive, which leads to misdetection of genes that are systematically
rearranged at low probability. The zero-inflated component of our model
accounts for this:
\begin{align}
p(Y_{ij} \mid M) = \begin{cases}
\kappa + (1 - \kappa) \operatorname{BB}\left(0 \mid N_{i}, \theta_{ij}, \phi
\right), & \text{if $Y_{ij}$ = 0} \
(1 - \kappa) \operatorname{BB}\left(Y_{ij} \mid N_{i}, \theta_{ij}, \phi
\right), & \text{if $Y_{ij}$ > 0}
\end{cases}\
\theta_{ij} = \operatorname{logit^{-1}}\left(\alpha_{j}+\beta_{ij}X_{i}\right)\
\beta_{ij}\sim\operatorname{Normal}\left(\gamma_{j},\gamma_{\sigma} \right)\
\gamma_{j}\sim\operatorname{Normal}\left(\hat{\gamma},\hat{\gamma}{\sigma}
\right) \
\alpha{j}\sim\operatorname{Normal}\left(\hat{\alpha},\hat{\alpha}{\sigma}
\right) \
\hat{\gamma} \sim \operatorname{Normal}\left(0, 5\right) \
\hat{\alpha} \sim \operatorname{Normal}\left(0, 10\right) \
\gamma{\sigma}, \hat{\gamma}{\sigma}, \hat{\alpha}{\sigma} \sim
\operatorname{Cauchy^{+}}\left(0, 1\right) \
\phi \sim \operatorname{Exponential}\left(\tau\right) \
\tau \sim \operatorname{Gamma}\left(3, 0.1\right) \
\kappa \sim \operatorname{Beta}\left(1, 3\right)
\end{align}
Model $M$ legend:
- $i$ and $j$: index of different repertoires and genes, respectively
- $\kappa$: zero-inflation probability
- $\theta$: probability of gene usage
- $\phi$: dispersion
- $\alpha$: intercept/baseline gene usage
- $\beta$: slope/within-repertoire DGU coefficient
- $\gamma$, $\gamma_{\sigma}$: slope/gene-specific DGU coefficient;
standard deviation
- $\hat{\gamma}$, $\hat{\gamma}_{\sigma}$: mean and standard deviation of
the population of gene-specific DGU coefficients
- $\hat{\alpha}$, $\hat{\alpha}_{\sigma}$: mean and standard deviation of
the population of gene-specific baseline usages
- $\operatorname{BB}$: beta-binomial probability mass function (pmf)
- $\operatorname{Normal}$: normal probability density function (pdf)
- $\operatorname{Cauchy^{+}}$: half-Cauchy pdf
- $\operatorname{Exponential}$: exponential pdf
- $\operatorname{Gamma}$: gamma pdf
- $\operatorname{Beta}$: beta pdf
- $\operatorname{logit^{-1}}$: inverse logistic function
In the output of IgGeneUsage, we report the mean effect size ($\gamma$) and its
95% highest density interval (HDI). Genes with $\gamma \neq 0$ (e.g. if 95% HDI
of $\gamma$ excludes 0) are most likely to experience differential usage.
Additionally, we report the probability of differential gene usage ($\pi$):
\begin{align}
\pi = 2 \cdot \max\left(\int_{\gamma = -\infty}^{0} p(\gamma)\mathrm{d}\gamma,
\int_{\gamma = 0}^{\infty} p(\gamma)\mathrm{d}\gamma\right) - 1
\end{align}
with $\pi = 1$ for genes with strong differential usage, and $\pi = 0$ for
genes with negligible differential gene usage. Both metrics are computed based
on the posterior distribution of $\gamma$, and are thus related. We find $\pi$
slightly easier to interpret.
Case Study I
IgGeneUsage provides built-in datasets from studies that evaluate biases in Ig
gene usage. The dataset IGHV_HCV contains publicly available data of human
immunoglobulin heavy chain VDJ rearrangements from a study that evaluates the
effect of HCV infection on the human BCR repertoire[^1]. The dataset consists
of a population of class-switched memory (CSM) B cells of 22 HCV-infected
patients (HCV+) and 7 healthy donors (HD). Gene usage data is available for 69
IGHV gene segments.
[^1]: Tucci, Felicia A., et al. "Biased IGH VDJ gene repertoire and clonal
expansions in B cells of chronically hepatitis C virus–infected individuals."
Blood 131.5 (2018): 546-557.]
Input data
The data is already formatted such that it can directly be passed as input to
IgGeneUsage. The following steps allow you to load the data, and to inspect
its column names and table entries.
data("IGHV_HCV", package = "IgGeneUsage")
kable(x = head(IGHV_HCV), row.names = FALSE)
Data visualization
Lets visualize the gene usage with ggplot2.
# we can compute the total number of rearrangements per sample
total.usage <- aggregate(gene_usage_count~sample_id+condition,
FUN = sum, data = IGHV_HCV)
total.usage$total <- total.usage$gene_usage_count
total.usage$gene_usage_count <- NULL
# merge it with the original data
viz <- merge(x = IGHV_HCV, y = total.usage,
by = c("sample_id", "condition"),
all.x = TRUE)
# compute %
viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100
# For this example lets only consider the top 45 genes
# Hint: In real analyses you should not filter any gene
top <- aggregate(gene_usage_pct~gene_name, data = viz, FUN = mean)
top <- top[order(top$gene_usage_pct, decreasing = TRUE), ]
IGHV_HCV <- IGHV_HCV[IGHV_HCV$gene_name %in% top$gene_name[seq_len(45)], ]
viz <- viz[viz$gene_name %in% top$gene_name[seq_len(45)], ]
# visualize
ggplot(data = viz)+
geom_point(aes(x = gene_name, y = gene_usage_pct,
fill = condition, shape = condition),
position = position_dodge(width = .7), stroke = 0)+
theme_bw(base_size = 11)+
ylab(label = "Usage [%]")+
xlab(label = '')+
theme(legend.position = "top")+
theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_shape_manual(name = "Condition", values = c(21, 22))
Analysis of differential gene usage with IgGeneUsage
This is the main method of IgGeneUsage. The primary input is the dataset
IGHV_HCV. Other inputs allow you to configure specific settings of the Markov
Chain Monte Carlo (MCMC) simulation.
In this example we analyze IGHV_HCV with 2 MCMC chains (750 iterations each,
including 250 warm-ups) in parallel using 2 CPU cores. We compute for each
parameter of our model its 95% highest density interval (HDIs).
Important remark: you should run your analysis using the default argument
values of the function DGU. These values have been carefully chosen to fit most
analyses for differential gene usage of immune repertoires. If any messages or
warnings are reported concerning the MCMC sampling, please consult the Stan
manual^2 and adjust the MCMC arguments accordingly. If the warnings persist,
please submit an issue with a reproducible script at the Bioconductor support
site or on Github^3.
M <- DGU(usage.data = IGHV_HCV, # input data
mcmc.warmup = 750, # how many MCMC warm-ups per chain (default: 500)
mcmc.steps = 2000, # how many MCMC steps per chain (default: 1,500)
mcmc.chains = 2, # how many MCMC chain to run (default: 4)
mcmc.cores = 1, # how many PC cores to use? (e.g. parallel chains)
hdi.level = 0.95, # highest density interval level (de fault: 0.95)
adapt.delta = 0.95, # MCMC target acceptance rate (default: 0.95)
max.treedepth = 10) # tree depth evaluated at each step (default: 12)
Output
The following objects are provided as part of the output of DGU:
- glm.summary (main results of IgGeneUsage): quantitative summary of
differential gene usage (see section 'Results')
- test.summary: alternative quantitative summary of differential gene usage
estimated with two frequentist methods (the Welch's t-test and the Wilcoxon
signed-rank test)
- glm: rstan ('stanfit') object of the fitted model $rightarrow$ you can use
this for further checks of your model (see section 'Model checking')
- ppc.data: data on posterior predictive checks (see section 'Model checking')
summary(M)
Model checking
Do not blindly trust your model $\rightarrow$ check it extensively. You can
use the object glm for these checks.
- Checklist of successful MCMC sampling^2:
- no divergences, no excessive warnings from rstan, Rhat < 1.05,
high Neff
- Checklist for valid model:
- observed data can be replicated with the fitted model $\rightarrow$
posterior predictive checks
- leave-one-out analysis
MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
gridExtra::grid.arrange(rstan::stan_rhat(object = M$glm),
rstan::stan_ess(object = M$glm),
nrow = 1)
Posterior predictive checks: repertoire-specific
IgGeneUsage has built-in checks for repertoire-specific posterior prediction
of gene usage. It uses the fitted model to predict the usage of each gene in
a given repertoire (y-axis). Error bars show 95% HDI of mean posterior
prediction. The predictions can be compared with the observed data (x-axis).
For points near the diagnoal $\rightarrow$ accurate prediction.
The following figure shows that our model can reproduce the fitted data
$\rightarrow$ this is an indicator of a valid model.
ggplot(data = M$ppc.data$ppc.repertoire)+
facet_wrap(facets = ~sample_name, ncol = 5)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_count, y = ppc_mean_count,
ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+
geom_point(aes(x = observed_count, y = ppc_mean_count,
fill = condition), shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_x_log10(breaks = c(1, 10, 100, 1000),
labels = expression(10^0, 10^1, 10^2, 10^3))+
scale_y_log10(breaks = c(1, 10, 100, 1000),
labels = expression(10^0, 10^1, 10^2, 10^3))+
xlab(label = "Observed usage [counts]")+
ylab(label = "Predicted usage [counts]")+
annotation_logticks(base = 10, sides = "lb")
Posterior predictive checks: overall
IgGeneUsage has built-in checks for gene-specific posterior prediction of gene
usage within each biological condition. We show the predictions (y-axis) of
the model, and compare them against the observed mean usage (x-axis). If the
points are near the diagnoal $\rightarrow$ accurate prediction. Errors show
95% HDI of mean posterior prediction.
ggplot(data = M$ppc.data$ppc.gene)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100), col = "darkgray")+
geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100,
fill = condition), shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
xlab(label = "Observed usage [%]")+
ylab(label = "Predicted usage [%]")+
scale_x_log10()+
scale_y_log10()+
annotation_logticks(base = 10, sides = "lb")
Results
Next we present the main results of IgGeneUsage contained in the data.frame
glm.summary. Each row of glm.summary summarizes the degree of differential
gene usage in a specific immune gene using two metrics (es and pmax).
Legend:
- es: effect size ($\gamma$ from model $M$) on differential gene usage
(mean, median standard error (se), standard deviation (sd), L (low boundary
of 95% HDI), H (high boundary of 95% HDI))
- contrast: the direction of the effect (e.g. tumor - healthy)
- pmax: probability of differential gene usage ($\pi$ from model $M$)
kable(x = head(M$glm.summary), row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown
as a point). Names are shown of genes with $\pi \geq 0.9$. Dashed horizontal
line represents null-effect ($\gamma = 0$). Notice that the gene with $\pi
\approx 1$ also has an effect size whose 95% HDI (error bar) does not overlap
the null-effect. The genes with high degree of differential usage are easy to
detect with this figure.
# format data
stats <- M$glm.summary
stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ]
stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name)
stats <- merge(x = stats, y = M$test.summary, by = "gene_name")
ggplot(data = stats)+
geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+
geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H),
col = "darkgray")+
geom_point(aes(x = pmax, y = es_mean))+
geom_text_repel(data = stats[stats$pmax >= 0.9, ],
aes(x = pmax, y = es_mean, label = gene_fac))+
theme_bw(base_size = 11)+
xlab(label = expression(pi))
Promising hits
Lets visualize the observed data of the genes with high probability of
differential gene usage ($\pi \geq 0.9$). Here we show the gene usage in %.
promising.genes <- stats$gene_name[stats$pmax >= 0.9]
ppc.gene <- M$ppc.data$ppc.gene
ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ]
ggplot()+
geom_errorbar(data = ppc.gene,
aes(x = gene_name, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100, col = condition),
position = position_dodge(width = .8), width = 0.75)+
geom_point(data = viz[viz$gene_name %in% promising.genes, ],
aes(x = gene_name, y = gene_usage_pct, col = condition),
shape = 21, size = 1.5, fill = "black",
position = position_jitterdodge(jitter.width = 0.15,
jitter.height = 0,
dodge.width = 0.8))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
ylab(label = "Usage [%]")+
xlab(label = '')
Promising hits [count]
Lets also visualize the gene usage frequencies. Point size represents
total usage in repertoire.
promising.genes <- stats$gene_name[stats$pmax >= 0.9]
ggplot(data = viz[viz$gene_name %in% promising.genes, ])+
facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+
geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition,
size = total/10^3), shape = 21)+
theme_bw(base_size = 11)+
ylab(label = "Usage [count]")+
xlab(label = '')+
theme(legend.position = "top")+
theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+
ylab(label = "Usage [count]")+
xlab(label = '')+
scale_y_log10()+
annotation_logticks(base = 10, sides = "l")
Comparison with the Welch's t-test (T-test)
Despite the fact that the data is not normaly distributed, we performed the
analysis of differential gene usage with the T-test. Prior to using the T-test,
we must convert the usage frequencies into proportions. This is automatically
done by IgGeneUsage (object test.summary).
Next, we compare the probabilities of differential gene usage ($\pi$) with the
FDR corrected P-values (-log10 scale) from the T-test. Dashed lines show
significance levels of 0.05 and 0.01. We observe few disagreements between the
two tests. Lets inspect them in more detail.
ggplot()+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(data = stats, col = "red", size = 2,
aes(x = pmax, y = -log10(t.test.fdr.pvalue)))+
geom_text_repel(data = stats[stats$pmax >= 0.5, ],
aes(x = pmax, y = -log10(t.test.fdr.pvalue),
label = gene_name), size = 4,
min.segment.length = 0.1)+
xlim(0, 1)+
ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Outliers: IGHV1-58 and IGHV3-72
The two genes IGHV1-58 and IGHV3-72 are prioritized by the T-test. Their $\pi$
estimates are however far from 1. Violation of T-test's assumptions is the most
probable cause for the disagreement. Discarding information about the sample
size, and thus about uncertainty could be an alternative explanation. Lets
visualize the data for IGHV1-58 and IGHV3-72.
promising.genes <- c("IGHV1-58", "IGHV3-72")
ppc.gene <- M$ppc.data$ppc.gene
ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ]
ggplot()+
geom_errorbar(data = ppc.gene,
aes(x = gene_name, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100, col = condition),
position = position_dodge(width = .8), width = 0.75)+
geom_point(data = viz[viz$gene_name %in% promising.genes, ],
aes(x = gene_name, y = gene_usage_pct, col = condition),
shape = 21, size = 1.5, fill = "black",
position = position_jitterdodge(jitter.width = 0.15,
jitter.height = 0,
dodge.width = 0.8))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
ylab(label = "Gene usage [%]")+
xlab(label = '')
Outliers: IGHV1-58 and IGHV3-72 [counts]
promising.genes <- c("IGHV1-58", "IGHV3-72")
ggplot(data = viz[viz$gene_name %in% promising.genes, ])+
facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+
geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition,
size = total/10^3), shape = 21)+
theme_bw(base_size = 11)+
ylab(label = "Usage [count]")+
xlab(label = '')+
theme(legend.position = "top")+
theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+
ylab(label = "Usage [count]")+
xlab(label = '')+
scale_y_log10()+
annotation_logticks(base = 10, sides = "l")
Comparison with the Wilcoxon signed-rank test (U-test)
The nonparametric U-test can also be used for the analysis of differential gene
usage. It assumes data with equal shape in both groups (also not met by our
data). Prior to using the U-test, we must again convert the usage frequencies
into proportions. This is automatically done by IgGeneUsage.
Lets also compare $\pi$ with the FDR corrected P-values (-log10 scale) from
the U-test. Dashed lines show significance levels of 0.05 and 0.01. The U-test
finds no evidence of differential gene usage.
ggplot()+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(data = stats, col = "red", size = 2,
aes(x = pmax, y = -log10(u.test.fdr.pvalue)))+
geom_text_repel(data = stats[stats$pmax >= 0.5, ],
aes(x = pmax, y = -log10(u.test.fdr.pvalue),
label = gene_name), size = 4,
min.segment.length = 0.1)+
xlim(0, 1)+
ylab(label = "-log10 (P-value) from U-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Case Study II
For our second case study we use a dataset from a study evaluating
vaccine-induced changes in B-cell populations [^5]. The data is publicly
provided via the R-package alakazam (version 0.2.11). The IGHV family usage is
reported in four B-cell populations (repertoires IgM, IgD, IgG and IgA) across
two timepoints (conditions = -1 hour vs. +7 days). In this case study we
investigate the overal effect of the vaccine on the IGHV family usage.
[^5]: Laserson U and Vigneault F, et al. High-resolution antibody dynamics of
vaccine-induced immune responses. Proc Natl Acad Sci USA. 2014 111:4928-33.
Input data
The data is already formatted such that it can directly be passed as input to
IgGeneUsage. Lets load the data, and to inspect its content.
data(Ig, package = "IgGeneUsage")
kable(x = head(Ig), row.names = FALSE)
Data visualization
Lets look at the gene usage data with ggplot2.
# we can compute the total number of rearrangements per sample
total.usage <- aggregate(gene_usage_count~sample_id+condition,
FUN = sum, data = Ig)
total.usage$total <- total.usage$gene_usage_count
total.usage$gene_usage_count <- NULL
# merge it with the original data
viz <- merge(x = Ig, y = total.usage,
by = c("sample_id", "condition"),
all.x = TRUE)
# compute %
viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100
# visualize
ggplot(data = viz)+
geom_point(aes(x = gene_name, y = gene_usage_pct, fill = condition,
shape = condition), stroke = 0, size = 3,
position = position_jitterdodge(jitter.width = 0.25,
dodge.width = 0.75))+
theme_bw(base_size = 11)+
ylab(label = "Usage [%]")+
xlab(label = '')+
theme(legend.position = "top")+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_shape_manual(name = "Condition", values = c(21, 22))
Analysis with IgGeneUsage
Lets analyze the differential gene usage in the data Ig. We use the
default values of most DGU arguments.
M <- DGU(usage.data = Ig,
mcmc.warmup = 500,
mcmc.steps = 1500,
mcmc.chains = 2,
mcmc.cores = 1,
hdi.level = 0.95,
adapt.delta = 0.95,
max.treedepth = 12)
Model checking
Checking MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
gridExtra::grid.arrange(rstan::stan_rhat(object = M$glm),
rstan::stan_ess(object = M$glm),
nrow = 1)
Posterior predictive checks: repertoire-specific
ggplot(data = M$ppc$ppc.repertoire)+
facet_wrap(facets = ~sample_name, ncol = 4)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_count, y = ppc_mean_count,
ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+
geom_point(aes(x = observed_count, y = ppc_mean_count,
fill = condition), shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_x_log10(breaks = c(1, 10, 100, 1000),
labels = expression(10^0, 10^1, 10^2, 10^3))+
scale_y_log10(breaks = c(1, 10, 100, 1000),
labels = expression(10^0, 10^1, 10^2, 10^3))+
xlab(label = "Observed usage [counts]")+
ylab(label = "Predicted usage [counts]")+
annotation_logticks(base = 10, sides = "bl")
Posterior predictive checks: overall
ggplot(data = M$ppc.data$ppc.gene)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100), col = "darkgray")+
geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100,
fill = condition), shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
xlab(label = "Observed usage [%]")+
ylab(label = "Predicted usage [%]")+
scale_x_log10()+
scale_y_log10()+
annotation_logticks(base = 10, sides = "bl")
Results
Main results:
kable(x = M$glm.summary, row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown
as a point). There are no genes with $\pi \approx 1$ $\rightarrow$ no sufficiently
strong evidence of differential gene usage based on IgGeneUsage.
# format data
stats <- M$glm.summary
stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ]
stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name)
stats <- merge(x= stats, y = M$test.summary, by = "gene_name")
ggplot(data = stats)+
geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+
geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H),
col = "darkgray")+
geom_point(aes(x = pmax, y = es_mean))+
geom_text_repel(data = stats, aes(x = pmax, y = es_mean, label = gene_fac))+
theme_bw(base_size = 11)+
xlab(label = expression(pi))+
xlim(0, 1)
Comparison with the Welch's t-test (T-test)
ggplot()+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(data = stats, col = "red", size = 2,
aes(x = pmax, y = -log10(t.test.fdr.pvalue)))+
geom_text_repel(data = stats,
aes(x = pmax, y = -log10(t.test.fdr.pvalue),
label = gene_name), size = 4,
min.segment.length = 0.1)+
xlim(0, 1)+
ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Outlier: IGHV5
promising.genes <- unique(M$usage.data$gene_names)
ppc.gene <- M$ppc.data$ppc.gene
ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ]
ggplot()+
geom_errorbar(data = ppc.gene,
aes(x = gene_name, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100, col = condition),
position = position_dodge(width = .8), width = 0.75)+
geom_point(data = viz[viz$gene_name %in% promising.genes, ],
aes(x = gene_name, y = gene_usage_pct, col = condition),
shape = 21, size = 1.5, fill = "black",
position = position_jitterdodge(jitter.width = 0.15,
jitter.height = 0,
dodge.width = 0.8))+
theme_bw(base_size = 11)+
theme(legend.position = "top")
Outliers IGHV5 (original data)
promising.genes <- "IGHV5"
ggplot(data = viz[viz$gene_name %in% promising.genes, ])+
facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+
geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition,
size = total/10^3), shape = 21)+
theme_bw(base_size = 11)+
ylab(label = "Usage [count]")+
xlab(label = '')+
theme(legend.position = "top")+
theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+
ylab(label = "Usage [count]")+
xlab(label = '')+
scale_y_log10()+
annotation_logticks(base = 10, sides = "l")
Comparison with the Wilcoxon signed-rank test (U-test)
No evidence of differential usage (at FDR level 0.05)
ggplot()+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(data = stats, col = "red", size = 2,
aes(x = pmax, y = -log10(u.test.fdr.pvalue)))+
geom_text_repel(data = stats,
aes(x = pmax, y = -log10(u.test.fdr.pvalue),
label = gene_name), size = 4,
min.segment.length = 0.1)+
xlim(0, 1)+
ylab(label = "-log10 (P-value) from U-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Case Study III
The main purpose of using IgGeneUsage is to discover differential gene usage.
However, the tool can be be used for all differential count analyses. In the
third case study we explain how to detect differential usage of net CDR3
sequence charge between repertoires belonging to two biological conditions. For
this purpose we use CDR3 sequence data of human T-cells (TRB-chain) downloaded
from VDJdb ^4.
Each CDR3 sequence is annotated to a specific viral epitope. Here we focus
on the two viruses InfluenzaA and CMV which represent the two biological
conditions. We consider the different data sources (publications) as different
repertoires. To compute the net sequence charge, we consider the amino acids
K, R and H as +1 charged, while D and E as -1 charged. Thus, we computed the
net charge of a CDR3 sequence by adding up the individual residue charges.
Input data
The data is already formatted such that it can directly be passed as input to
IgGeneUsage. Lets load the data, and to inspect its content.
data(CDR3_Epitopes, package = "IgGeneUsage")
kable(x = head(CDR3_Epitopes), row.names = FALSE)
Data visualization
Lets look at the input data with ggplot2.
# we can compute the total number of rearrangements per sample
total.usage <- aggregate(gene_usage_count~sample_id+condition,
FUN = sum, data = CDR3_Epitopes)
total.usage$total <- total.usage$gene_usage_count
total.usage$gene_usage_count <- NULL
# merge it with the original data
viz <- merge(x = CDR3_Epitopes, y = total.usage,
by = c("sample_id", "condition"),
all.x = TRUE)
# compute %
viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100
# visualize
ggplot(data = viz)+
geom_point(aes(x = as.numeric(gene_name),
y = gene_usage_pct, fill = condition,
shape = condition), stroke = 0, size = 2, alpha = 0.5,
position = position_jitterdodge(jitter.width = 0.15,
dodge.width = 0.5))+
theme_bw(base_size = 11)+
ylab(label = "Usage [%]")+
xlab(label = 'Net Charge')+
theme(legend.position = "top")+
scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+
scale_shape_manual(name = "Condition", values = c(21, 22))+
scale_x_continuous(breaks = -7:7, labels = -7:7)
Analysis with IgGeneUsage
Here we study differential gene usage in the data CDR3_Epitopes. We use the
default values of most DGU arguments.
M <- DGU(usage.data = CDR3_Epitopes,
mcmc.chains = 2,
mcmc.cores = 1)
# default DGU parameters:
# mcmc.warmup = 500
# mcmc.steps = 1,500
# mcmc.chains = 2
# mcmc.cores = 1
# hdi.level = 0.95
# adapt.delta = 0.95
# max.treedepth = 12
Model checking
MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
grid.arrange(rstan::stan_rhat(object = M$glm),
rstan::stan_ess(object = M$glm),
nrow = 1)
Posterior predictive checks: repertoire-specific
ggplot(data = M$ppc.data$ppc.repertoire)+
facet_wrap(facets = ~sample_name, ncol = 4)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_count, y = ppc_mean_count,
ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+
geom_point(aes(x = observed_count, y = ppc_mean_count, fill = condition),
shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
xlab(label = "Observed usage [counts]")+
ylab(label = "Predicted usage [counts]")+
scale_x_log10()+
scale_y_log10()+
annotation_logticks(base = 10, sides = "bl")
Posterior predictive checks: overall differential usage
ggplot(data = M$ppc.data$ppc.gene)+
geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+
geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100), col = "darkgray")+
geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100,
fill = condition), shape = 21, size = 1)+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
xlab(label = "Observed usage [%]")+
ylab(label = "Predicted usage [%]")+
scale_x_log10()+
scale_y_log10()+
annotation_logticks(base = 10, sides = "bl")
Results
Main results:
kable(x = head(M$glm.summary), row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown
as a point). There are couple of net-charges groups with $\pi \approx 1$.
# format data
stats <- M$glm.summary
stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ]
stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name)
stats <- merge(x = stats, y = M$test.summary, by = "gene_name")
ggplot(data = stats)+
geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+
geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H),
col = "darkgray")+
geom_point(aes(x = pmax, y = es_mean))+
geom_text_repel(data = stats,
aes(x = pmax, y = es_mean, label = gene_fac))+
theme_bw(base_size = 11)+
xlab(label = expression(pi))+
scale_x_continuous(limits = c(0, 1))
Comparison with the Welch's t-test
ggplot(data = stats)+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(aes(x = pmax, y = -log10(t.test.fdr.pvalue)),
size = 1.5, col = "red")+
geom_text_repel(aes(x = pmax, y = -log10(t.test.fdr.pvalue),
label = gene_name), size = 5)+
xlim(0, 1)+
ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Comparison with the Wilcoxon signed-rank test (U-test)
ggplot(data = stats)+
geom_hline(yintercept = c(-log10(0.05), -log10(0.01)),
linetype = "dashed", col = "darkgray")+
geom_point(aes(x = pmax, y = -log10(u.test.fdr.pvalue)),
size = 1.5, col = "red")+
geom_text_repel(aes(x = pmax, y = -log10(u.test.fdr.pvalue),
label = gene_name), size = 5)+
xlim(0, 1)+
ylab(label = "-log10(p-value) from U-test [FDR corrected]")+
xlab(label = expression(pi))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
scale_color_discrete(name = '')
Net charge usage [in %] groups and posterior means (and 95% HDI of means)
promising.genes <- M$usage.data$gene_names
ppc.gene <- M$ppc.data$ppc.gene
ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ]
ppc.gene$gene_name <- factor(x = ppc.gene$gene_name, levels = -5:6)
ggplot()+
geom_errorbar(data = ppc.gene,
aes(x = gene_name, ymin = ppc_L_prop*100,
ymax = ppc_H_prop*100, col = condition),
position = position_dodge(width = .8), width = 0.75)+
geom_point(data = viz[viz$gene_name %in% promising.genes, ],
aes(x = gene_name, y = gene_usage_pct, col = condition),
shape = 21, size = 1.5, fill = "black",
position = position_jitterdodge(jitter.width = 0.2,
jitter.height = 0,
dodge.width = 0.75))+
theme_bw(base_size = 11)+
theme(legend.position = "top")+
ylab(label = "Gene usage [%]")+
xlab(label = '')
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IgGeneUsage documentation built on Nov. 8, 2020, 7:47 p.m.
R Package Documentation
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Note that we can't provide technical support on individual packages. You should contact the package authors for that.
knitr::opts_chunk$set(comment = FALSE, warning = FALSE, message = FALSE)
require(IgGeneUsage) require(knitr) require(ggplot2) require(ggforce) require(gridExtra) require(ggrepel) require(rstan) require(reshape2) rstan_options(auto_write = TRUE)
Introduction
Decoding the properties of immune repertoires is key in understanding the response of adaptive immunity to challenges such as viral infection. One important property is biases in Ig gene usage between biological conditions (e.g. healthy vs turmor). Yet, most analyses for differential gene usage are performed qualitatively, or with inadequate statistical methods. Here we introduce IgGeneUsage, a computational tool for the analysis of differential gene usage.
Input
The input to IgGeneUsage is a data.frame object with usage frequencies for each gene of a repertoire that belongs to a particular biological condition. The usage data.frame has the following 4 columns:
- sample_id: identifier of the repertoire (e.g. Patient-1)
- condition: identifier of the condition to which each repertoire belongs (e.g. healthy or tumor)
- gene_name: specific gene name (e.g. IGHV1-10 or family TRVB1)
- gene_usage_count: numeric (count) of usage related to columns 1-3
The sum of all gene usage counts (column 4) for a given repertoire should be equal to the total gene usage in that repertoire.
Method
IgGeneUsage transforms the provided input in the following way. Given $R$ repertoires, each having $G$ genes, IgGeneUsage generates a gene usage matrix $Y^{R \times G}$. Row sums in $Y$ define the total usage in each repertoire ($N$). The design variable $X$ is set to $X = 1$ for repertoires that belong to the first condition, and $X = -1$ otherwise.
For the analysis of DGU between two biological conditions, we designed the following Bayesian model ($M$) for zero-inflated beta-binomial regression. This model can fit over-dispersed gene usage data. The immune repertoire data is also not exhaustive, which leads to misdetection of genes that are systematically rearranged at low probability. The zero-inflated component of our model accounts for this:
\begin{align} p(Y_{ij} \mid M) = \begin{cases} \kappa + (1 - \kappa) \operatorname{BB}\left(0 \mid N_{i}, \theta_{ij}, \phi \right), & \text{if $Y_{ij}$ = 0} \ (1 - \kappa) \operatorname{BB}\left(Y_{ij} \mid N_{i}, \theta_{ij}, \phi \right), & \text{if $Y_{ij}$ > 0} \end{cases}\ \theta_{ij} = \operatorname{logit^{-1}}\left(\alpha_{j}+\beta_{ij}X_{i}\right)\ \beta_{ij}\sim\operatorname{Normal}\left(\gamma_{j},\gamma_{\sigma} \right)\ \gamma_{j}\sim\operatorname{Normal}\left(\hat{\gamma},\hat{\gamma}{\sigma} \right) \ \alpha{j}\sim\operatorname{Normal}\left(\hat{\alpha},\hat{\alpha}{\sigma} \right) \ \hat{\gamma} \sim \operatorname{Normal}\left(0, 5\right) \ \hat{\alpha} \sim \operatorname{Normal}\left(0, 10\right) \ \gamma{\sigma}, \hat{\gamma}{\sigma}, \hat{\alpha}{\sigma} \sim \operatorname{Cauchy^{+}}\left(0, 1\right) \ \phi \sim \operatorname{Exponential}\left(\tau\right) \ \tau \sim \operatorname{Gamma}\left(3, 0.1\right) \ \kappa \sim \operatorname{Beta}\left(1, 3\right) \end{align}
Model $M$ legend:
- $i$ and $j$: index of different repertoires and genes, respectively
- $\kappa$: zero-inflation probability
- $\theta$: probability of gene usage
- $\phi$: dispersion
- $\alpha$: intercept/baseline gene usage
- $\beta$: slope/within-repertoire DGU coefficient
- $\gamma$, $\gamma_{\sigma}$: slope/gene-specific DGU coefficient; standard deviation
- $\hat{\gamma}$, $\hat{\gamma}_{\sigma}$: mean and standard deviation of the population of gene-specific DGU coefficients
- $\hat{\alpha}$, $\hat{\alpha}_{\sigma}$: mean and standard deviation of the population of gene-specific baseline usages
- $\operatorname{BB}$: beta-binomial probability mass function (pmf)
- $\operatorname{Normal}$: normal probability density function (pdf)
- $\operatorname{Cauchy^{+}}$: half-Cauchy pdf
- $\operatorname{Exponential}$: exponential pdf
- $\operatorname{Gamma}$: gamma pdf
- $\operatorname{Beta}$: beta pdf
- $\operatorname{logit^{-1}}$: inverse logistic function
In the output of IgGeneUsage, we report the mean effect size ($\gamma$) and its 95% highest density interval (HDI). Genes with $\gamma \neq 0$ (e.g. if 95% HDI of $\gamma$ excludes 0) are most likely to experience differential usage. Additionally, we report the probability of differential gene usage ($\pi$): \begin{align} \pi = 2 \cdot \max\left(\int_{\gamma = -\infty}^{0} p(\gamma)\mathrm{d}\gamma, \int_{\gamma = 0}^{\infty} p(\gamma)\mathrm{d}\gamma\right) - 1 \end{align} with $\pi = 1$ for genes with strong differential usage, and $\pi = 0$ for genes with negligible differential gene usage. Both metrics are computed based on the posterior distribution of $\gamma$, and are thus related. We find $\pi$ slightly easier to interpret.
Case Study I
IgGeneUsage provides built-in datasets from studies that evaluate biases in Ig gene usage. The dataset IGHV_HCV contains publicly available data of human immunoglobulin heavy chain VDJ rearrangements from a study that evaluates the effect of HCV infection on the human BCR repertoire[^1]. The dataset consists of a population of class-switched memory (CSM) B cells of 22 HCV-infected patients (HCV+) and 7 healthy donors (HD). Gene usage data is available for 69 IGHV gene segments.
[^1]: Tucci, Felicia A., et al. "Biased IGH VDJ gene repertoire and clonal expansions in B cells of chronically hepatitis C virus–infected individuals." Blood 131.5 (2018): 546-557.]
Input data
The data is already formatted such that it can directly be passed as input to IgGeneUsage. The following steps allow you to load the data, and to inspect its column names and table entries.
data("IGHV_HCV", package = "IgGeneUsage")
kable(x = head(IGHV_HCV), row.names = FALSE)
Data visualization
Lets visualize the gene usage with ggplot2.
# we can compute the total number of rearrangements per sample total.usage <- aggregate(gene_usage_count~sample_id+condition, FUN = sum, data = IGHV_HCV) total.usage$total <- total.usage$gene_usage_count total.usage$gene_usage_count <- NULL # merge it with the original data viz <- merge(x = IGHV_HCV, y = total.usage, by = c("sample_id", "condition"), all.x = TRUE) # compute % viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100 # For this example lets only consider the top 45 genes # Hint: In real analyses you should not filter any gene top <- aggregate(gene_usage_pct~gene_name, data = viz, FUN = mean) top <- top[order(top$gene_usage_pct, decreasing = TRUE), ] IGHV_HCV <- IGHV_HCV[IGHV_HCV$gene_name %in% top$gene_name[seq_len(45)], ] viz <- viz[viz$gene_name %in% top$gene_name[seq_len(45)], ] # visualize ggplot(data = viz)+ geom_point(aes(x = gene_name, y = gene_usage_pct, fill = condition, shape = condition), position = position_dodge(width = .7), stroke = 0)+ theme_bw(base_size = 11)+ ylab(label = "Usage [%]")+ xlab(label = '')+ theme(legend.position = "top")+ theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_shape_manual(name = "Condition", values = c(21, 22))
Analysis of differential gene usage with IgGeneUsage
This is the main method of IgGeneUsage. The primary input is the dataset IGHV_HCV. Other inputs allow you to configure specific settings of the Markov Chain Monte Carlo (MCMC) simulation.
In this example we analyze IGHV_HCV with 2 MCMC chains (750 iterations each, including 250 warm-ups) in parallel using 2 CPU cores. We compute for each parameter of our model its 95% highest density interval (HDIs).
Important remark: you should run your analysis using the default argument values of the function DGU. These values have been carefully chosen to fit most analyses for differential gene usage of immune repertoires. If any messages or warnings are reported concerning the MCMC sampling, please consult the Stan manual^2 and adjust the MCMC arguments accordingly. If the warnings persist, please submit an issue with a reproducible script at the Bioconductor support site or on Github^3.
M <- DGU(usage.data = IGHV_HCV, # input data mcmc.warmup = 750, # how many MCMC warm-ups per chain (default: 500) mcmc.steps = 2000, # how many MCMC steps per chain (default: 1,500) mcmc.chains = 2, # how many MCMC chain to run (default: 4) mcmc.cores = 1, # how many PC cores to use? (e.g. parallel chains) hdi.level = 0.95, # highest density interval level (de fault: 0.95) adapt.delta = 0.95, # MCMC target acceptance rate (default: 0.95) max.treedepth = 10) # tree depth evaluated at each step (default: 12)
Output
The following objects are provided as part of the output of DGU:
- glm.summary (main results of IgGeneUsage): quantitative summary of differential gene usage (see section 'Results')
- test.summary: alternative quantitative summary of differential gene usage estimated with two frequentist methods (the Welch's t-test and the Wilcoxon signed-rank test)
- glm: rstan ('stanfit') object of the fitted model $rightarrow$ you can use this for further checks of your model (see section 'Model checking')
- ppc.data: data on posterior predictive checks (see section 'Model checking')
summary(M)
Model checking
Do not blindly trust your model $\rightarrow$ check it extensively. You can use the object glm for these checks.
- Checklist of successful MCMC sampling^2:
- no divergences, no excessive warnings from rstan, Rhat < 1.05, high Neff
- Checklist for valid model:
- observed data can be replicated with the fitted model $\rightarrow$ posterior predictive checks
- leave-one-out analysis
MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
gridExtra::grid.arrange(rstan::stan_rhat(object = M$glm), rstan::stan_ess(object = M$glm), nrow = 1)
Posterior predictive checks: repertoire-specific
IgGeneUsage has built-in checks for repertoire-specific posterior prediction of gene usage. It uses the fitted model to predict the usage of each gene in a given repertoire (y-axis). Error bars show 95% HDI of mean posterior prediction. The predictions can be compared with the observed data (x-axis). For points near the diagnoal $\rightarrow$ accurate prediction.
The following figure shows that our model can reproduce the fitted data $\rightarrow$ this is an indicator of a valid model.
ggplot(data = M$ppc.data$ppc.repertoire)+ facet_wrap(facets = ~sample_name, ncol = 5)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_count, y = ppc_mean_count, ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+ geom_point(aes(x = observed_count, y = ppc_mean_count, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_x_log10(breaks = c(1, 10, 100, 1000), labels = expression(10^0, 10^1, 10^2, 10^3))+ scale_y_log10(breaks = c(1, 10, 100, 1000), labels = expression(10^0, 10^1, 10^2, 10^3))+ xlab(label = "Observed usage [counts]")+ ylab(label = "Predicted usage [counts]")+ annotation_logticks(base = 10, sides = "lb")
Posterior predictive checks: overall
IgGeneUsage has built-in checks for gene-specific posterior prediction of gene usage within each biological condition. We show the predictions (y-axis) of the model, and compare them against the observed mean usage (x-axis). If the points are near the diagnoal $\rightarrow$ accurate prediction. Errors show 95% HDI of mean posterior prediction.
ggplot(data = M$ppc.data$ppc.gene)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100), col = "darkgray")+ geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ xlab(label = "Observed usage [%]")+ ylab(label = "Predicted usage [%]")+ scale_x_log10()+ scale_y_log10()+ annotation_logticks(base = 10, sides = "lb")
Results
Next we present the main results of IgGeneUsage contained in the data.frame glm.summary. Each row of glm.summary summarizes the degree of differential gene usage in a specific immune gene using two metrics (es and pmax).
Legend:
- es: effect size ($\gamma$ from model $M$) on differential gene usage (mean, median standard error (se), standard deviation (sd), L (low boundary of 95% HDI), H (high boundary of 95% HDI))
- contrast: the direction of the effect (e.g. tumor - healthy)
- pmax: probability of differential gene usage ($\pi$ from model $M$)
kable(x = head(M$glm.summary), row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown as a point). Names are shown of genes with $\pi \geq 0.9$. Dashed horizontal line represents null-effect ($\gamma = 0$). Notice that the gene with $\pi \approx 1$ also has an effect size whose 95% HDI (error bar) does not overlap the null-effect. The genes with high degree of differential usage are easy to detect with this figure.
# format data stats <- M$glm.summary stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ] stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name) stats <- merge(x = stats, y = M$test.summary, by = "gene_name") ggplot(data = stats)+ geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+ geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H), col = "darkgray")+ geom_point(aes(x = pmax, y = es_mean))+ geom_text_repel(data = stats[stats$pmax >= 0.9, ], aes(x = pmax, y = es_mean, label = gene_fac))+ theme_bw(base_size = 11)+ xlab(label = expression(pi))
Promising hits
Lets visualize the observed data of the genes with high probability of differential gene usage ($\pi \geq 0.9$). Here we show the gene usage in %.
promising.genes <- stats$gene_name[stats$pmax >= 0.9] ppc.gene <- M$ppc.data$ppc.gene ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ] ggplot()+ geom_errorbar(data = ppc.gene, aes(x = gene_name, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100, col = condition), position = position_dodge(width = .8), width = 0.75)+ geom_point(data = viz[viz$gene_name %in% promising.genes, ], aes(x = gene_name, y = gene_usage_pct, col = condition), shape = 21, size = 1.5, fill = "black", position = position_jitterdodge(jitter.width = 0.15, jitter.height = 0, dodge.width = 0.8))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ ylab(label = "Usage [%]")+ xlab(label = '')
Promising hits [count]
Lets also visualize the gene usage frequencies. Point size represents total usage in repertoire.
promising.genes <- stats$gene_name[stats$pmax >= 0.9] ggplot(data = viz[viz$gene_name %in% promising.genes, ])+ facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+ geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition, size = total/10^3), shape = 21)+ theme_bw(base_size = 11)+ ylab(label = "Usage [count]")+ xlab(label = '')+ theme(legend.position = "top")+ theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+ ylab(label = "Usage [count]")+ xlab(label = '')+ scale_y_log10()+ annotation_logticks(base = 10, sides = "l")
Comparison with the Welch's t-test (T-test)
Despite the fact that the data is not normaly distributed, we performed the analysis of differential gene usage with the T-test. Prior to using the T-test, we must convert the usage frequencies into proportions. This is automatically done by IgGeneUsage (object test.summary).
Next, we compare the probabilities of differential gene usage ($\pi$) with the FDR corrected P-values (-log10 scale) from the T-test. Dashed lines show significance levels of 0.05 and 0.01. We observe few disagreements between the two tests. Lets inspect them in more detail.
ggplot()+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(data = stats, col = "red", size = 2, aes(x = pmax, y = -log10(t.test.fdr.pvalue)))+ geom_text_repel(data = stats[stats$pmax >= 0.5, ], aes(x = pmax, y = -log10(t.test.fdr.pvalue), label = gene_name), size = 4, min.segment.length = 0.1)+ xlim(0, 1)+ ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Outliers: IGHV1-58 and IGHV3-72
The two genes IGHV1-58 and IGHV3-72 are prioritized by the T-test. Their $\pi$ estimates are however far from 1. Violation of T-test's assumptions is the most probable cause for the disagreement. Discarding information about the sample size, and thus about uncertainty could be an alternative explanation. Lets visualize the data for IGHV1-58 and IGHV3-72.
promising.genes <- c("IGHV1-58", "IGHV3-72") ppc.gene <- M$ppc.data$ppc.gene ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ] ggplot()+ geom_errorbar(data = ppc.gene, aes(x = gene_name, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100, col = condition), position = position_dodge(width = .8), width = 0.75)+ geom_point(data = viz[viz$gene_name %in% promising.genes, ], aes(x = gene_name, y = gene_usage_pct, col = condition), shape = 21, size = 1.5, fill = "black", position = position_jitterdodge(jitter.width = 0.15, jitter.height = 0, dodge.width = 0.8))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ ylab(label = "Gene usage [%]")+ xlab(label = '')
Outliers: IGHV1-58 and IGHV3-72 [counts]
promising.genes <- c("IGHV1-58", "IGHV3-72") ggplot(data = viz[viz$gene_name %in% promising.genes, ])+ facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+ geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition, size = total/10^3), shape = 21)+ theme_bw(base_size = 11)+ ylab(label = "Usage [count]")+ xlab(label = '')+ theme(legend.position = "top")+ theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+ ylab(label = "Usage [count]")+ xlab(label = '')+ scale_y_log10()+ annotation_logticks(base = 10, sides = "l")
Comparison with the Wilcoxon signed-rank test (U-test)
The nonparametric U-test can also be used for the analysis of differential gene usage. It assumes data with equal shape in both groups (also not met by our data). Prior to using the U-test, we must again convert the usage frequencies into proportions. This is automatically done by IgGeneUsage.
Lets also compare $\pi$ with the FDR corrected P-values (-log10 scale) from the U-test. Dashed lines show significance levels of 0.05 and 0.01. The U-test finds no evidence of differential gene usage.
ggplot()+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(data = stats, col = "red", size = 2, aes(x = pmax, y = -log10(u.test.fdr.pvalue)))+ geom_text_repel(data = stats[stats$pmax >= 0.5, ], aes(x = pmax, y = -log10(u.test.fdr.pvalue), label = gene_name), size = 4, min.segment.length = 0.1)+ xlim(0, 1)+ ylab(label = "-log10 (P-value) from U-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Case Study II
For our second case study we use a dataset from a study evaluating vaccine-induced changes in B-cell populations [^5]. The data is publicly provided via the R-package alakazam (version 0.2.11). The IGHV family usage is reported in four B-cell populations (repertoires IgM, IgD, IgG and IgA) across two timepoints (conditions = -1 hour vs. +7 days). In this case study we investigate the overal effect of the vaccine on the IGHV family usage.
[^5]: Laserson U and Vigneault F, et al. High-resolution antibody dynamics of vaccine-induced immune responses. Proc Natl Acad Sci USA. 2014 111:4928-33.
Input data
The data is already formatted such that it can directly be passed as input to IgGeneUsage. Lets load the data, and to inspect its content.
data(Ig, package = "IgGeneUsage")
kable(x = head(Ig), row.names = FALSE)
Data visualization
Lets look at the gene usage data with ggplot2.
# we can compute the total number of rearrangements per sample total.usage <- aggregate(gene_usage_count~sample_id+condition, FUN = sum, data = Ig) total.usage$total <- total.usage$gene_usage_count total.usage$gene_usage_count <- NULL # merge it with the original data viz <- merge(x = Ig, y = total.usage, by = c("sample_id", "condition"), all.x = TRUE) # compute % viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100 # visualize ggplot(data = viz)+ geom_point(aes(x = gene_name, y = gene_usage_pct, fill = condition, shape = condition), stroke = 0, size = 3, position = position_jitterdodge(jitter.width = 0.25, dodge.width = 0.75))+ theme_bw(base_size = 11)+ ylab(label = "Usage [%]")+ xlab(label = '')+ theme(legend.position = "top")+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_shape_manual(name = "Condition", values = c(21, 22))
Analysis with IgGeneUsage
Lets analyze the differential gene usage in the data Ig. We use the default values of most DGU arguments.
M <- DGU(usage.data = Ig, mcmc.warmup = 500, mcmc.steps = 1500, mcmc.chains = 2, mcmc.cores = 1, hdi.level = 0.95, adapt.delta = 0.95, max.treedepth = 12)
Model checking
Checking MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
gridExtra::grid.arrange(rstan::stan_rhat(object = M$glm), rstan::stan_ess(object = M$glm), nrow = 1)
Posterior predictive checks: repertoire-specific
ggplot(data = M$ppc$ppc.repertoire)+ facet_wrap(facets = ~sample_name, ncol = 4)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_count, y = ppc_mean_count, ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+ geom_point(aes(x = observed_count, y = ppc_mean_count, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_x_log10(breaks = c(1, 10, 100, 1000), labels = expression(10^0, 10^1, 10^2, 10^3))+ scale_y_log10(breaks = c(1, 10, 100, 1000), labels = expression(10^0, 10^1, 10^2, 10^3))+ xlab(label = "Observed usage [counts]")+ ylab(label = "Predicted usage [counts]")+ annotation_logticks(base = 10, sides = "bl")
Posterior predictive checks: overall
ggplot(data = M$ppc.data$ppc.gene)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100), col = "darkgray")+ geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ xlab(label = "Observed usage [%]")+ ylab(label = "Predicted usage [%]")+ scale_x_log10()+ scale_y_log10()+ annotation_logticks(base = 10, sides = "bl")
Results
Main results:
kable(x = M$glm.summary, row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown as a point). There are no genes with $\pi \approx 1$ $\rightarrow$ no sufficiently strong evidence of differential gene usage based on IgGeneUsage.
# format data stats <- M$glm.summary stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ] stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name) stats <- merge(x= stats, y = M$test.summary, by = "gene_name") ggplot(data = stats)+ geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+ geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H), col = "darkgray")+ geom_point(aes(x = pmax, y = es_mean))+ geom_text_repel(data = stats, aes(x = pmax, y = es_mean, label = gene_fac))+ theme_bw(base_size = 11)+ xlab(label = expression(pi))+ xlim(0, 1)
Comparison with the Welch's t-test (T-test)
ggplot()+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(data = stats, col = "red", size = 2, aes(x = pmax, y = -log10(t.test.fdr.pvalue)))+ geom_text_repel(data = stats, aes(x = pmax, y = -log10(t.test.fdr.pvalue), label = gene_name), size = 4, min.segment.length = 0.1)+ xlim(0, 1)+ ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Outlier: IGHV5
promising.genes <- unique(M$usage.data$gene_names) ppc.gene <- M$ppc.data$ppc.gene ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ] ggplot()+ geom_errorbar(data = ppc.gene, aes(x = gene_name, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100, col = condition), position = position_dodge(width = .8), width = 0.75)+ geom_point(data = viz[viz$gene_name %in% promising.genes, ], aes(x = gene_name, y = gene_usage_pct, col = condition), shape = 21, size = 1.5, fill = "black", position = position_jitterdodge(jitter.width = 0.15, jitter.height = 0, dodge.width = 0.8))+ theme_bw(base_size = 11)+ theme(legend.position = "top")
Outliers IGHV5 (original data)
promising.genes <- "IGHV5" ggplot(data = viz[viz$gene_name %in% promising.genes, ])+ facet_wrap(facets = ~gene_name, ncol = 1, scales = "free_y")+ geom_point(aes(x = sample_id, y = gene_usage_count, fill = condition, size = total/10^3), shape = 21)+ theme_bw(base_size = 11)+ ylab(label = "Usage [count]")+ xlab(label = '')+ theme(legend.position = "top")+ theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.4))+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_size_continuous(name = expression("N ("*10^3*")"), range = c(1, 5))+ ylab(label = "Usage [count]")+ xlab(label = '')+ scale_y_log10()+ annotation_logticks(base = 10, sides = "l")
Comparison with the Wilcoxon signed-rank test (U-test)
No evidence of differential usage (at FDR level 0.05)
ggplot()+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(data = stats, col = "red", size = 2, aes(x = pmax, y = -log10(u.test.fdr.pvalue)))+ geom_text_repel(data = stats, aes(x = pmax, y = -log10(u.test.fdr.pvalue), label = gene_name), size = 4, min.segment.length = 0.1)+ xlim(0, 1)+ ylab(label = "-log10 (P-value) from U-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Case Study III
The main purpose of using IgGeneUsage is to discover differential gene usage. However, the tool can be be used for all differential count analyses. In the third case study we explain how to detect differential usage of net CDR3 sequence charge between repertoires belonging to two biological conditions. For this purpose we use CDR3 sequence data of human T-cells (TRB-chain) downloaded from VDJdb ^4.
Each CDR3 sequence is annotated to a specific viral epitope. Here we focus on the two viruses InfluenzaA and CMV which represent the two biological conditions. We consider the different data sources (publications) as different repertoires. To compute the net sequence charge, we consider the amino acids K, R and H as +1 charged, while D and E as -1 charged. Thus, we computed the net charge of a CDR3 sequence by adding up the individual residue charges.
Input data
The data is already formatted such that it can directly be passed as input to IgGeneUsage. Lets load the data, and to inspect its content.
data(CDR3_Epitopes, package = "IgGeneUsage")
kable(x = head(CDR3_Epitopes), row.names = FALSE)
Data visualization
Lets look at the input data with ggplot2.
# we can compute the total number of rearrangements per sample total.usage <- aggregate(gene_usage_count~sample_id+condition, FUN = sum, data = CDR3_Epitopes) total.usage$total <- total.usage$gene_usage_count total.usage$gene_usage_count <- NULL # merge it with the original data viz <- merge(x = CDR3_Epitopes, y = total.usage, by = c("sample_id", "condition"), all.x = TRUE) # compute % viz$gene_usage_pct <- viz$gene_usage_count/viz$total*100 # visualize ggplot(data = viz)+ geom_point(aes(x = as.numeric(gene_name), y = gene_usage_pct, fill = condition, shape = condition), stroke = 0, size = 2, alpha = 0.5, position = position_jitterdodge(jitter.width = 0.15, dodge.width = 0.5))+ theme_bw(base_size = 11)+ ylab(label = "Usage [%]")+ xlab(label = 'Net Charge')+ theme(legend.position = "top")+ scale_fill_manual(name = "Condition", values = c("orange", "#6666ff"))+ scale_shape_manual(name = "Condition", values = c(21, 22))+ scale_x_continuous(breaks = -7:7, labels = -7:7)
Analysis with IgGeneUsage
Here we study differential gene usage in the data CDR3_Epitopes. We use the default values of most DGU arguments.
M <- DGU(usage.data = CDR3_Epitopes, mcmc.chains = 2, mcmc.cores = 1) # default DGU parameters: # mcmc.warmup = 500 # mcmc.steps = 1,500 # mcmc.chains = 2 # mcmc.cores = 1 # hdi.level = 0.95 # adapt.delta = 0.95 # max.treedepth = 12
Model checking
MCMC sampling
- divergences, tree-depth, energy
rstan::check_hmc_diagnostics(M$glm)
- Rhat and Neff
grid.arrange(rstan::stan_rhat(object = M$glm), rstan::stan_ess(object = M$glm), nrow = 1)
Posterior predictive checks: repertoire-specific
ggplot(data = M$ppc.data$ppc.repertoire)+ facet_wrap(facets = ~sample_name, ncol = 4)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_count, y = ppc_mean_count, ymin = ppc_L_count, ymax = ppc_H_count), col = "darkgray")+ geom_point(aes(x = observed_count, y = ppc_mean_count, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ xlab(label = "Observed usage [counts]")+ ylab(label = "Predicted usage [counts]")+ scale_x_log10()+ scale_y_log10()+ annotation_logticks(base = 10, sides = "bl")
Posterior predictive checks: overall differential usage
ggplot(data = M$ppc.data$ppc.gene)+ geom_abline(intercept = 0, slope = 1, linetype = "dashed", col = "darkgray")+ geom_errorbar(aes(x = observed_prop*100, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100), col = "darkgray")+ geom_point(aes(x = observed_prop*100, y = ppc_mean_prop*100, fill = condition), shape = 21, size = 1)+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ xlab(label = "Observed usage [%]")+ ylab(label = "Predicted usage [%]")+ scale_x_log10()+ scale_y_log10()+ annotation_logticks(base = 10, sides = "bl")
Results
Main results:
kable(x = head(M$glm.summary), row.names = FALSE, digits = 3)
The effect size and $\pi$ are related. Lets visualize them for each gene (shown as a point). There are couple of net-charges groups with $\pi \approx 1$.
# format data stats <- M$glm.summary stats <- stats[order(abs(stats$es_mean), decreasing = FALSE), ] stats$gene_fac <- factor(x = stats$gene_name, levels = stats$gene_name) stats <- merge(x = stats, y = M$test.summary, by = "gene_name") ggplot(data = stats)+ geom_hline(yintercept = 0, linetype = "dashed", col = "gray")+ geom_errorbar(aes(x = pmax, y = es_mean, ymin = es_L, ymax = es_H), col = "darkgray")+ geom_point(aes(x = pmax, y = es_mean))+ geom_text_repel(data = stats, aes(x = pmax, y = es_mean, label = gene_fac))+ theme_bw(base_size = 11)+ xlab(label = expression(pi))+ scale_x_continuous(limits = c(0, 1))
Comparison with the Welch's t-test
ggplot(data = stats)+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(aes(x = pmax, y = -log10(t.test.fdr.pvalue)), size = 1.5, col = "red")+ geom_text_repel(aes(x = pmax, y = -log10(t.test.fdr.pvalue), label = gene_name), size = 5)+ xlim(0, 1)+ ylab(label = "-log10 (P-value) from t-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Comparison with the Wilcoxon signed-rank test (U-test)
ggplot(data = stats)+ geom_hline(yintercept = c(-log10(0.05), -log10(0.01)), linetype = "dashed", col = "darkgray")+ geom_point(aes(x = pmax, y = -log10(u.test.fdr.pvalue)), size = 1.5, col = "red")+ geom_text_repel(aes(x = pmax, y = -log10(u.test.fdr.pvalue), label = gene_name), size = 5)+ xlim(0, 1)+ ylab(label = "-log10(p-value) from U-test [FDR corrected]")+ xlab(label = expression(pi))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ scale_color_discrete(name = '')
Net charge usage [in %] groups and posterior means (and 95% HDI of means)
promising.genes <- M$usage.data$gene_names ppc.gene <- M$ppc.data$ppc.gene ppc.gene <- ppc.gene[ppc.gene$gene_name %in% promising.genes, ] ppc.gene$gene_name <- factor(x = ppc.gene$gene_name, levels = -5:6) ggplot()+ geom_errorbar(data = ppc.gene, aes(x = gene_name, ymin = ppc_L_prop*100, ymax = ppc_H_prop*100, col = condition), position = position_dodge(width = .8), width = 0.75)+ geom_point(data = viz[viz$gene_name %in% promising.genes, ], aes(x = gene_name, y = gene_usage_pct, col = condition), shape = 21, size = 1.5, fill = "black", position = position_jitterdodge(jitter.width = 0.2, jitter.height = 0, dodge.width = 0.75))+ theme_bw(base_size = 11)+ theme(legend.position = "top")+ ylab(label = "Gene usage [%]")+ xlab(label = '')
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