GGPUBR: How to Add P-Values Generated Elsewhere to a GGPLOT

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05 May

GGPUBR: How to Add P-Values Generated Elsewhere to a GGPLOT

Alboukadel

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This article describes how to add p-values generated elsewhere to a ggplot using the ggpubr package. The following key ggpubr functions will be used:

  • stat_pvalue_manual(): Add manually p-values to a ggplot, such as box blots, dot plots and stripcharts.
  • geom_bracket(): Add brackets with label annotation to a ggplot. Helpers for adding p-value or significance levels to a plot.

You will learn how to:

  • Add custom p-values created from elsewhere
  • Add p-values obtained from the rstatix R package
  • Add brackets with custom p-value labels to a ggplot


Contents:

Prerequisites

Make sure you have the following R packages:

  • tidyverse for data manipulation and visualization
  • ggpubr for creating easily publication ready plots
  • rstatix provides pipe-friendly R functions for easy statistical analyses

Load required R packages:

library(tidyverse)
library(ggpubr)
library(rstatix)

Data preparation

# Convert `dose` variable into factor
df <- ToothGrowth
df$dose <- factor(df$dose)

# Inspect the data 
# Display two random rows by groups
set.seed(123)
df %>% sample_n_by(dose, size = 2)
## # A tibble: 6 x 3
##     len supp  dose 
##   <dbl> <fct> <fct>
## 1  10   VC    0.5  
## 2  14.5 OJ    0.5  
## 3  18.8 VC    1    
## 4  25.8 OJ    1    
## 5  29.4 OJ    2    
## 6  23.6 VC    2

Add p-values computed from elsewhere

Key R function: stat_pvalue_manual() [in ggpubr package]

stat_pvalue_manual(data, label = NULL)
  • data: a data frame containing statistical test results. The expected default format should contain the following columns: group1 | group2 | p | y.position | etc. group1 and group2 are the groups that have been compared. p is the resulting p-value. y.position is the y coordinates of the p-values in the plot.
  • label: the column containing the label (e.g.: label = "p" or label = "p.adj"), where p is the p-value. Can be also an expression that can be formatted by the glue package. For example, when specifying label = "t-test, p = {p}", the expression {p} will be replaced by its value.
# p-values
stat.test <- tibble::tribble(
  ~group1, ~group2,   ~p.adj,
    "0.5",     "1", 2.54e-07,
    "0.5",     "2", 1.32e-13,
      "1",     "2", 1.91e-05
  )
stat.test
## # A tibble: 3 x 3
##   group1 group2    p.adj
##   <chr>  <chr>     <dbl>
## 1 0.5    1      2.54e- 7
## 2 0.5    2      1.32e-13
## 3 1      2      1.91e- 5
# Box plots + p-values
ggboxplot(df, x = "dose", y = "len") +
  stat_pvalue_manual(
    stat.test, 
    y.position = 35, step.increase = 0.1,
    label = "p.adj"
    )

Add p-values obtained from the rstatix package

Create a simple box plot

bxp <- ggboxplot(df, x = "dose", y = "len")
bxp

Pairwise comparisons

# Statistical test
stat.test <- df %>% t_test(len ~ dose)
stat.test
## # A tibble: 3 x 10
##   .y.   group1 group2    n1    n2 statistic    df        p    p.adj p.adj.signif
## * <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>    <dbl>    <dbl> <chr>       
## 1 len   0.5    1         20    20     -6.48  38.0 1.27e- 7 2.54e- 7 ****        
## 2 len   0.5    2         20    20    -11.8   36.9 4.40e-14 1.32e-13 ****        
## 3 len   1      2         20    20     -4.90  37.1 1.91e- 5 1.91e- 5 ****
# Box plot
stat.test <- stat.test %>% add_xy_position(x = "dose")
bxp + stat_pvalue_manual(stat.test, label = "p.adj.signif", tip.length = 0.01)

Faceted plots

# Statistical tests
stat.test <- df %>%
  group_by(supp) %>%
  t_test(len ~ dose, ref.group = "0.5")
stat.test
## # A tibble: 4 x 11
##   supp  .y.   group1 group2    n1    n2 statistic    df            p        p.adj p.adj.signif
## * <fct> <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>        <dbl>        <dbl> <chr>       
## 1 OJ    len   0.5    1         10    10     -5.05  17.7 0.0000878    0.0000878    ****        
## 2 OJ    len   0.5    2         10    10     -7.82  14.7 0.00000132   0.00000264   ****        
## 3 VC    len   0.5    1         10    10     -7.46  17.9 0.000000681  0.000000681  ****        
## 4 VC    len   0.5    2         10    10    -10.4   14.3 0.0000000468 0.0000000936 ****
# Box plots
stat.test <- stat.test %>% add_xy_position(x = "dose")
ggboxplot(df, x = "dose", y = "len", facet.by = "supp") +
  stat_pvalue_manual(stat.test, label = "p.adj.signif", tip.length = 0.01)

Grouped plots

# Box plot: comparison against reference
stat.test <- df %>%
  group_by(supp) %>%
  t_test(len ~ dose, ref.group = "0.5") 

# Box plots
stat.test <- stat.test %>% 
  add_xy_position(x = "supp", dodge = 0.8)
bxp <- ggboxplot(df, x = "supp", y = "len", color = "dose")
bxp + stat_pvalue_manual(stat.test,   label = "p.adj", tip.length = 0.01)

Specify manually the y position of the p-values

Create a simple box plot:

# Pairwise t-test between groups
stat.test <- ToothGrowth %>%
  t_test(len ~ dose) %>%
  mutate(y.position = c(29, 35, 39))
stat.test
## # A tibble: 3 x 11
##   .y.   group1 group2    n1    n2 statistic    df        p    p.adj p.adj.signif y.position
## * <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>    <dbl>    <dbl> <chr>             <dbl>
## 1 len   0.5    1         20    20     -6.48  38.0 1.27e- 7 2.54e- 7 ****                 29
## 2 len   0.5    2         20    20    -11.8   36.9 4.40e-14 1.32e-13 ****                 35
## 3 len   1      2         20    20     -4.90  37.1 1.91e- 5 1.91e- 5 ****                 39
# Create a box plot and add the p-value
ggboxplot(ToothGrowth, x = "dose", y = "len") +
  stat_pvalue_manual(stat.test, label = "p.adj")

Faceted plots: Comparisons between two groups

# Pairwise t-test between groups
stat.test <- df %>%
  group_by(dose) %>%
  t_test(len ~ supp) %>%
  adjust_pvalue() %>%
  mutate(y.position = 35)
stat.test
## # A tibble: 3 x 11
##   dose  .y.   group1 group2    n1    n2 statistic    df       p   p.adj y.position
## * <fct> <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>   <dbl>   <dbl>      <dbl>
## 1 0.5   len   OJ     VC        10    10    3.17    15.0 0.00636 0.0127          35
## 2 1     len   OJ     VC        10    10    4.03    15.4 0.00104 0.00312         35
## 3 2     len   OJ     VC        10    10   -0.0461  14.0 0.964   0.964           35
# Create a box plot and add the p-value
p <- ggboxplot(
  df, x = "supp", y = "len",
   color = "supp", palette = "jco",
  facet.by = "dose", ylim = c(0, 40)
  )

p + stat_pvalue_manual(stat.test, label = "p.adj")

Faceted plots: Pairwise comparisons between multiple groups

# Pairwise t-test between groups
stat.test <- df %>%
  group_by(supp) %>%
  t_test(len ~ dose) %>%
  adjust_pvalue() %>%
  mutate(y.position = rep(c(29, 35, 39), 2))
stat.test
## # A tibble: 6 x 12
##   supp  .y.   group1 group2    n1    n2 statistic    df            p       p.adj p.adj.signif y.position
## * <fct> <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>        <dbl>       <dbl> <chr>             <dbl>
## 1 OJ    len   0.5    1         10    10     -5.05  17.7 0.0000878    0.000263    ***                  29
## 2 OJ    len   0.5    2         10    10     -7.82  14.7 0.00000132   0.00000528  ****                 35
## 3 OJ    len   1      2         10    10     -2.25  15.8 0.039        0.039       *                    39
## 4 VC    len   0.5    1         10    10     -7.46  17.9 0.000000681  0.00000340  ****                 29
## 5 VC    len   0.5    2         10    10    -10.4   14.3 0.0000000468 0.000000281 ****                 35
## 6 VC    len   1      2         10    10     -5.47  13.6 0.0000916    0.000263    ****                 39
# Create a box plot and add the p-value
p <- ggboxplot(
  df, x = "dose", y = "len",
   color = "supp", palette = "jco",
  facet.by = "supp", ylim = c(0, 40)
  )

p + stat_pvalue_manual(stat.test)

Grouped plots

# Pairwise t-test between groups
stat.test <- df %>%
  group_by(dose) %>%
  t_test(len ~ supp) %>%
  adjust_pvalue() %>%
  mutate(y.position = 35)
stat.test
## # A tibble: 3 x 11
##   dose  .y.   group1 group2    n1    n2 statistic    df       p   p.adj y.position
## * <fct> <chr> <chr>  <chr>  <int> <int>     <dbl> <dbl>   <dbl>   <dbl>      <dbl>
## 1 0.5   len   OJ     VC        10    10    3.17    15.0 0.00636 0.0127          35
## 2 1     len   OJ     VC        10    10    4.03    15.4 0.00104 0.00312         35
## 3 2     len   OJ     VC        10    10   -0.0461  14.0 0.964   0.964           35
# Create a box plot and add the p-value
p <- ggboxplot(
  ToothGrowth, x = "dose", y = "len",
   color = "supp", palette = "jco",
   ylim = c(0, 40)
  )

p + stat_pvalue_manual(stat.test, xmin = "dose", xmax = NULL)

Add brackets with custom p-value labels to a ggplot

This section describes the function geom_bracket() [in ggpubr package] for adding brackets with label annotation to a ggplot. It makes it easy to add p-value or significance levels created elsewhere to a plot.

Basic brackets with labels

# Add bracket with labels
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    xmin = "0.5", xmax = "1", y.position = 30,
    label = "t-test, p < 0.05"
  )

# Customize bracket tip.length tip.length
# Move up the label using vjust
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    xmin = "0.5", xmax = "1", y.position = 30,
    label = "t-test, p < 0.05", 
    tip.length = c(0.2, 0.02), vjust = -1
  )

Using plotmath expression and specifying multiple brackets manually

#Using plotmath expression
ggboxplot(df, x = "dose", y = "len") +
 geom_bracket(
   xmin = "0.5", xmax = "1", y.position = 30,
   label = "list(~italic(p)<=0.001)", type = "expression",
   tip.length = c(0.2, 0.02)
 )


# Specify multiple brackets manually
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    xmin = c("0.5", "1"), xmax = c("1", "2"),
    y.position = c(30, 35), label = c("***", "**"),
    tip.length = 0.01
  )

Compute statistical tests and add p-values

# Compute statistical tests and add p-values
stat.test <- compare_means(len ~ dose, ToothGrowth, method = "t.test")
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    aes(xmin = group1, xmax = group2, label = signif(p, 2)),
    data = stat.test, y.position = 35
  )

# Increase step length between brackets
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    aes(xmin = group1, xmax = group2, label = signif(p, 2)),
    data = stat.test, y.position = 35, step.increase = 0.1
  )

# Or specify the positions of each comparison
ggboxplot(df, x = "dose", y = "len") +
  geom_bracket(
    aes(xmin = group1, xmax = group2, label = signif(p, 2)),
    data = stat.test, y.position = c(32, 35, 38)
   )

Conclusions

This article describes how to add p-values generated elsewhere to a ggplot using the ggpubr package.



 
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Comments ( 4 )

  • Xiaochi Liu
    01 Jun 2020

    Hello Kassambara,

    Many thanks for your tutorial. It’s very helpful.

    In the document, “step.increase” argument means numeric vector with the increase in fraction of total height for every additional comparison to minimize overlap; and “tip.length” means numeric vector with the fraction of total height that the bar goes down to indicate the precise column.

    I was wondering, what is the “total height” in these two arguments’ definition? Could you please explain more about it?

    Many thanks for your help!

    Reply
    • Kassambara
      02 Jun 2020

      Thank you Xiaochi Liu for your positive feedback. The total height corresponds to the y-axis scale range (ymax-ymin)

      Reply
  • Luana M Simao
    20 Apr 2021

    What would it be the numbers on the function “mutate(y.position = rep(c…”?

    Reply
  • Gustavo
    11 Jun 2021

    Dear Kassambara,

    Great tutorial, thanks for sharing!

    I’d like to use stat_pvalue_manual() to include emmeans posthoc tests to my analysis. However, it doesn’t work the same way you described because emmeans produces a emmGrid as a result, and not a table like the t-test() here.

    I noticed that there is a function that is based on rstatix called emmeans_test. This one produces a table so I guess it could be used instead of emmeans. How similar would emmeans and emmeans_test be? And do you know by any chance why I get the following error message when I try to run emmeans_test?

    > df %>% emmeans_test(len ~ dose, p.adjust.method = “bonferroni”)
    Error: Column name `std.error` must not be duplicated.
    Use .name_repair to specify repair.

    Many thanks!! Cheers, Gustavo

    Reply

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