Figure 4 Analysis
Last updated: 2026-01-06
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Knit directory: DXR_website/
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Modified: analysis/DXR_Project_Analysis.Rmd
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| Rmd | cd535e9 | emmapfort | 2026-01-06 | Final Website Touches 01/06/25 EMP |
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| Rmd | 49fa865 | emmapfort | 2026-01-05 | Final Updates to Website EMP 01/05/26 |
| Rmd | da1f69c | emmapfort | 2025-12-02 | updated analysis 12/02/25 |
Libraries
Load necessary libraries for analysis.
library(tidyverse)
library(readxl)
library(readr)Define my custom plot theme
# Define the custom theme
# plot_theme_custom <- function() {
# theme_minimal() +
# theme(
# #line for x and y axis
# axis.line = element_line(linewidth = 1,
# color = "black"),
#
# #axis ticks only on x and y, length standard
# axis.ticks.x = element_line(color = "black",
# linewidth = 1),
# axis.ticks.y = element_line(color = "black",
# linewidth = 1),
# axis.ticks.length = unit(0.05, "in"),
#
# #text and font
# axis.text = element_text(color = "black",
# family = "Arial",
# size = 7),
# axis.title = element_text(color = "black",
# family = "Arial",
# size = 9),
# legend.text = element_text(color = "black",
# family = "Arial",
# size = 7),
# legend.title = element_text(color = "black",
# family = "Arial",
# size = 9),
# plot.title = element_text(color = "black",
# family = "Arial",
# size = 10),
#
# #blank background and border
# panel.background = element_blank(),
# panel.border = element_blank(),
#
# #gridlines for alignment
# panel.grid.major = element_line(color = "grey80", linewidth = 0.5), #grey major grid for align in illus
# panel.grid.minor = element_line(color = "grey90", linewidth = 0.5) #grey minor grid for align in illus
# )
# }
# saveRDS(plot_theme_custom, "data/plot_theme_custom.RDS")
theme_custom <- readRDS("data/plot_theme_custom.RDS")Define a function to save plots as .pdf and .png
save_plot <- function(plot, filename,
folder = ".",
width = 8,
height = 6,
units = "in",
dpi = 300,
add_date = TRUE) {
if (missing(filename)) stop("Please provide a filename (without extension) for the plot.")
date_str <- if (add_date) paste0("_", format(Sys.Date(), "%y%m%d")) else ""
pdf_file <- file.path(folder, paste0(filename, date_str, ".pdf"))
png_file <- file.path(folder, paste0(filename, date_str, ".png"))
ggsave(filename = pdf_file, plot = plot, device = cairo_pdf, width = width, height = height, units = units, bg = "transparent")
ggsave(filename = png_file, plot = plot, device = "png", width = width, height = height, units = units, dpi = dpi, bg = "transparent")
message("Saved plot as Cairo PDF: ", pdf_file)
message("Saved plot as PNG: ", png_file)
}
output_folder <- "C:/Users/emmap/OneDrive/Desktop/DXR Manuscript Materials/Fig pdfs"
#save plot function created
#to use: just define the plot name, filename_base, width, heightCompare Response Clusters to Known TOP2i Treatments
Read in TOP2i DEGs from Matthews et al., Paul et al.
Matthews ER, Johnson OD, Horn KJ, Gutiérrez JA, Powell SR, et al. (2024) Anthracyclines induce cardiotoxicity through a shared gene expression response signature. PLOS Genetics 20(2): e1011164. https://doi.org/10.1371/journal.pgen.1011164
Topoisomerase II inhibitors CX-5461 and Doxorubicin differ in their cardiotoxicity profiles Sayan Paul, José A. Gutiérrez, Alyssa R. Bogar, Michelle C. Ward. bioRxiv 2025.07.20.665626; doi: https://doi.org/10.1101/2025.07.20.665626
#These gene lists were obtained from:
#Matthews ER, Johnson OD, Horn KJ, Gutiérrez JA, Powell SR, et al. (2024) Anthracyclines induce cardiotoxicity through a shared gene expression response signature. PLOS Genetics 20(2): e1011164. https://doi.org/10.1371/journal.pgen.1011164
DEGs_RM_list <- readRDS("data/Fig4/DEGs_TOP2i_RM.RDS")
#only keep the conditions that I want to compare to
keep_conditions <- c("sigVDA24", "sigVDX24", "sigVEP24", "sigVMT24")
DEGs_RM_24 <- DEGs_RM_list[keep_conditions]
#change the first column to Entrez_ID for all sets
DEGs_RM_24 <- lapply(DEGs_RM_24, function(df) {
# Rename the first column to Entrez_ID, regardless of current name
colnames(df)[1] <- "Entrez_ID"
return(df)
})
#DOX
DOX_RM_DEGs <- DEGs_RM_24$sigVDX24 %>%
as.data.frame()
dim(DOX_RM_DEGs)[1] 6645 8#6645 genes
#DNR
DNR_RM_DEGs <- DEGs_RM_24$sigVDA24 %>%
as.data.frame()
dim(DNR_RM_DEGs)[1] 7017 8#7017 genes
#EPI
EPI_RM_DEGs <- DEGs_RM_24$sigVEP24 %>%
as.data.frame()
dim(EPI_RM_DEGs)[1] 6328 8#6328 genes
#MTX
MTX_RM_DEGs <- DEGs_RM_24$sigVMT24 %>%
as.data.frame()
dim(MTX_RM_DEGs)[1] 1115 8#1115 genes
#now combine these all into one dataframe for later
DEGs_RM_24_df <- dplyr::bind_rows(
# DOX_RM_DEGs %>% mutate(Drug = "DOX_RM"),
DNR_RM_DEGs %>% mutate(Drug = "DAUN_RM"),
EPI_RM_DEGs %>% mutate(Drug = "EPI_RM"),
MTX_RM_DEGs %>% mutate(Drug = "MITO_RM"),
)
#These gene lists were obtained from:
#Topoisomerase II inhibitors CX-5461 and Doxorubicin differ in their cardiotoxicity profiles
#Sayan Paul, José A. Gutiérrez, Alyssa R. Bogar, Michelle C. Ward
#bioRxiv 2025.07.20.665626; doi: https://doi.org/10.1101/2025.07.20.665626
#CX 0.5 24hr
CX_SP_24 <- read.csv("data/Fig4/Toptable_CX_0.5_24.csv")
dim(CX_SP_24)[1] 14279 7#14279 genes, make sure to now filter this for significance
#DOX 0.5 24hr
DOX_SP_24 <- read.csv("data/Fig4/Toptable_DOX_0.5_24.csv")
dim(DOX_SP_24)[1] 14279 7#14279 genes, filter for significance
#filter both above for adj.P.Val < 0.05
DEGs_SP_sig <- list(
"CX_SP" = CX_SP_24$Entrez_ID[CX_SP_24$adj.P.Val < 0.05],
"DOX_SP" = DOX_SP_24$Entrez_ID[DOX_SP_24$adj.P.Val < 0.05]
)
#now make dataframes and place them with RM's data to complete my Top2i comparative dataframe
CX_SP_DEGs <- CX_SP_24 %>%
filter(adj.P.Val < 0.05) %>%
as.data.frame()
dim(CX_SP_DEGs)[1] 278 7#278 genes
DOX_SP_DEGs <- DOX_SP_24 %>%
filter(adj.P.Val < 0.05) %>%
as.data.frame()
dim(DOX_SP_DEGs)[1] 9828 7#9828 genes
#since SP's data has Entrez_ID as an integer rather than a character, coerce these to character in order for bind_rows to work properly
# CX_SP_DEGs$Entrez_ID <- as.character(CX_SP_DEGs$Entrez_ID)
# DOX_SP_DEGs$Entrez_ID <- as.character(DOX_SP_DEGs$Entrez_ID)
#these two also don't have a SYMBOL column so let's add that in
#CX df
# CX_SP_DEGs <- CX_SP_DEGs %>%
# mutate(
# SYMBOL = mapIds(
# org.Hs.eg.db,
# keys = Entrez_ID,
# column = "SYMBOL",
# keytype = "ENTREZID",
# multiVals = "first"
# )
# ) %>%
# dplyr::select(Entrez_ID, SYMBOL, everything()) # move SYMBOL to second column
# Same for df6
# DOX_SP_DEGs <- DOX_SP_DEGs %>%
# mutate(
# SYMBOL = mapIds(
# org.Hs.eg.db,
# keys = Entrez_ID,
# column = "SYMBOL",
# keytype = "ENTREZID",
# multiVals = "first"
# )
# ) %>%
# dplyr::select(Entrez_ID, SYMBOL, everything())
# DEGs_Top2i_24_df <- dplyr::bind_rows(
# DOX_RM_DEGs %>% mutate(Treatment = "DOX_RM"),
# DNR_RM_DEGs %>% mutate(Treatment = "DAUN_RM"),
# EPI_RM_DEGs %>% mutate(Treatment = "EPI_RM"),
# MTX_RM_DEGs %>% mutate(Treatment = "MITO_RM"),
# CX_SP_DEGs %>% mutate(Treatment = "CX_SP"),
# DOX_SP_DEGs %>% mutate(Treatment = "DOX_SP")
# )
#now this table has all columns consistently named and in the same formats
# saveRDS(DEGs_Top2i_24_df, "data/Fig4/DEGlist_Top2i_SP_RM.RDS")
DEGs_Top2i_24_df <- readRDS("data/Fig4/DEGlist_Top2i_SP_RM.RDS")
#also make a list version just in case
# DEGs_Top2i_24_list <- list(
# "DOX_RM" = DOX_RM_DEGs, #6645 DEGs
# "DNR_RM" = DNR_RM_DEGs, #7017 DEGs
# "EPI_RM" = EPI_RM_DEGs, #6328 DEGs
# "MTX_RM" = MTX_RM_DEGs, #1115 DEGs
# "CX_SP" = CX_SP_DEGs, #278 DEGs
# "DOX_SP" = DOX_SP_DEGs #9828 DEGs
# )
#total 10,106 DEGs from SP
#total 21,105 DEGs from RM
# saveRDS(DEGs_Top2i_24_list, "data/Fig4/DEGs_Top2i_RM_SP_list.RDS")
DEGs_Top2i_24_list <- readRDS("data/Fig4/DEGs_Top2i_RM_SP_list.RDS")Figure 4A - Percentage of TOP2i DEGs in tx+0 DEGs
Proportion barplots with permutation testing to ascertain whether DEGs shared between DOX and other TOP2i treatments are more likely than chance to be in both groups.
#read in my DEGs
DOX_24T_R <- read.csv("data/DE/Toptable_RUV_24T_final.csv") %>% dplyr::select(-X)
DOX_24R_R <- read.csv("data/DE/Toptable_RUV_24R_final.csv") %>% dplyr::select(-X)
DOX_144R_R <- read.csv("data/DE/Toptable_RUV_144R_final.csv") %>% dplyr::select(-X)
deg_list <- list(
"t0" = DOX_24T_R$Entrez_ID[DOX_24T_R$adj.P.Val < 0.05],
"t24" = DOX_24R_R$Entrez_ID[DOX_24R_R$adj.P.Val < 0.05],
"t144" = DOX_144R_R$Entrez_ID[DOX_144R_R$adj.P.Val < 0.05]
)
#reference set - t0
t0_set <- unique(na.omit(as.character(deg_list$"t0")))
cat("Total t0 DEGs:", length(t0_set), "\n")Total t0 DEGs: 9420 #background set - all genes tested
all_genes <- readRDS("data/DE/all_genes.RDS")
cat("Total background genes:", length(all_genes), "\n")Total background genes: 14319 #define TOP2i treatment DEG sets from literature
comparison_DEGs <- list(
DOX_RM = as.character(DOX_RM_DEGs$Entrez_ID),
DNR_RM = as.character(DNR_RM_DEGs$Entrez_ID),
EPI_RM = as.character(EPI_RM_DEGs$Entrez_ID),
MTX_RM = as.character(MTX_RM_DEGs$Entrez_ID),
CX_SP = as.character(CX_SP_DEGs$Entrez_ID),
DOX_SP = as.character(DOX_SP_DEGs$Entrez_ID)
)
#build data for a stacked barplot with percentages
plot_df <- purrr::map_dfr(names(comparison_DEGs), function(name) {
comp_DEGs <- unique(comparison_DEGs[[name]])
overlap <- sum(t0_set %in% comp_DEGs)
non_overlap <- length(t0_set) - overlap
tibble(
Treatment = name,
Category = c("DEG", "nonDEG"),
Count = c(overlap, non_overlap),
Total = length(t0_set),
Fraction = c(overlap, non_overlap)/length(t0_set)
)
})
plot_df <- plot_df %>%
mutate(
Treatment = factor(Treatment, levels = names(comparison_DEGs)),
Category = factor(Category, levels = c("DEG", "nonDEG"))
)
#legend labels (with n counts)
legend_labels <- plot_df %>%
filter(Category == "DEG Status") %>%
mutate(Label = paste0(Category, " (n=", Count, ")")) %>%
dplyr::select(Treatment, Label) %>%
deframe()
#perform permutation testing for enrichment
perm_test_overlap <- function(gene_set, t0_genes, gene_universe, n_perm = 10000) {
observed <- length(intersect(t0_genes, gene_set))
perm_overlaps <- replicate(n_perm, {
random_genes <- sample(gene_universe, length(t0_genes))
length(intersect(random_genes, gene_set))
})
p_val <- mean(perm_overlaps >= observed)
enrichment <- observed / mean(perm_overlaps)
return(list(observed = observed,
mean_perm = mean(perm_overlaps),
enrichment = enrichment,
p_value = p_val))
}
perm_results <- lapply(comparison_DEGs,
perm_test_overlap,
t0_genes = t0_set,
gene_universe = all_genes,
n_perm = 10000)
#summary stats table
perm_summary_df <- map_dfr(names(perm_results), function(name) {
res <- perm_results[[name]]
tibble(
Treatment = name,
Observed = res$observed,
MeanPerm = res$mean_perm,
Enrichment = res$enrichment,
PValue = res$p_value
)
}) %>%
mutate(FDR = p.adjust(PValue, method = "BH"),
Significant = case_when(
FDR < 0.001 ~ "***",
FDR < 0.01 ~ "**",
FDR < 0.05 ~ "*",
TRUE ~ "ns"
))
#show summary stats table
print(perm_summary_df)# A tibble: 6 × 7
Treatment Observed MeanPerm Enrichment PValue FDR Significant
<chr> <int> <dbl> <dbl> <dbl> <dbl> <chr>
1 DOX_RM 4888 4302. 1.14 0 0 ***
2 DNR_RM 5138 4540. 1.13 0 0 ***
3 EPI_RM 4946 4093. 1.21 0 0 ***
4 MTX_RM 757 718. 1.05 0.0055 0.0066 **
5 CX_SP 162 180. 0.902 0.989 0.989 ns
6 DOX_SP 7902 6397. 1.24 0 0 *** #label positions
#inside overlap bars: percentages
overlap_labels <- plot_df %>%
filter(Category == "Overlap") %>%
left_join(perm_summary_df %>% dplyr::select(Treatment, Significant), by = "Treatment") %>%
mutate(
Label = paste0(round(Fraction*100, 1), ""),
y_pos = 0.5 #fixed position at 50%
)
#above bars: significance stars
label_positions <- plot_df %>%
group_by(Treatment) %>%
summarise(y_pos = 1.0, .groups = "drop") %>%
left_join(perm_summary_df %>% dplyr::select(Treatment, Significant), by = "Treatment")
#plot final barplot
TOP2i_Combined <- ggplot(plot_df, aes(x = Treatment, y = Fraction, fill = Category)) +
geom_bar(stat = "identity", position = "stack", width = 0.6) +
#percentages inside overlap bars (fixed position)
geom_text(
data = overlap_labels,
aes(x = Treatment, y = y_pos, label = Label),
inherit.aes = FALSE,
color = "white",
fontface = "bold",
size = 4
) +
#significance stars above bars
geom_text(
data = label_positions,
aes(x = Treatment, y = y_pos, label = Significant),
inherit.aes = FALSE,
size = 4,
fontface = "bold",
color = "black"
) +
scale_y_continuous(labels = c(0, 100)) +
scale_fill_manual(values = c("Yes" = "#e41a1c", "No" = "#377eb8"),
labels = legend_labels) +
labs(
title = "Proportion of TOP2i DEGs in tx+0 DEGs",
x = "TOP2i Treatment",
y = "Percentage of DOX tx+0 DEGs (%)",
fill = "TOP2i DEGs"
) +
theme_custom()
# save_plot(
# plot = TOP2i_Combined,
# folder = output_folder,
# filename = "TOP2i_Barplot_Combined_Fig4_EMP",
# height = 6,
# width = 8
# )Supplementary Figure 7 - TOP2i DEGs Acute vs Chronic Clusters
#read in my chronic and acute cluster gene lists
final_genes_1_RUV <- readRDS("data/Fig3/final_genes_1_RUV.RDS")
final_genes_2_RUV <- readRDS("data/Fig3/final_genes_2_RUV.RDS")
acute_set <- unique(na.omit(final_genes_1_RUV))
chronic_set <- unique(na.omit(final_genes_2_RUV))
all_genes [1] "653635" "729737" "102723897" "100132287" "102465432" "100133331"
[7] "79854" "643837" "100130417" "148398" "26155" "339451"
[13] "57801" "9636" "375790" "54991" "51150" "126792"
[19] "388581" "118424" "6339" "116983" "126789" "54973"
[25] "80772" "83756" "1855" "54587" "54998" "81669"
[31] "148413" "55052" "64856" "219293" "83858" "55210"
[37] "339453" "29101" "643988" "142678" "8510" "984"
[43] "728661" "8511" "728642" "9906" "65220" "2782"
[49] "5590" "100506504" "199990" "6497" "79906" "100129534"
[55] "11079" "5192" "55229" "127281" "440556" "63976"
[61] "27237" "1953" "127262" "49856" "57212" "148870"
[67] "388588" "57470" "9731" "1677" "339448" "261734"
[73] "8514" "26038" "6146" "388591" "23463" "387509"
[79] "11332" "83715" "8718" "57449" "79707" "3104"
[85] "9903" "148479" "90326" "55735" "23261" "9341"
[91] "8863" "3604" "11315" "54206" "50651" "473"
[97] "2023" "80045" "9563" "80176" "84275" "199953"
[103] "5293" "22883" "56998" "84328" "64802" "10277"
[109] "23095" "5226" "378708" "1676" "5195" "54897"
[115] "148345" "23435" "10747" "6723" "5394" "2475"
[121] "29914" "26232" "93611" "26270" "10459" "57085"
[127] "4524" "1185" "4878" "4879" "90231" "5351"
[133] "9927" "60672" "55187" "9249" "126755" "10630"
[139] "7799" "23254" "55092" "79180" "842" "23341"
[145] "84301" "6248" "23207" "284723" "54751" "440567"
[151] "729614" "23013" "7709" "27129" "1187" "1188"
[157] "348487" "1969" "128272" "79363" "54455" "26099"
[163] "55707" "114819" "55672" "84809" "11209" "11223"
[169] "9696" "4237" "23400" "6390" "11240" "55920"
[175] "55160" "127707" "8659" "126917" "23352" "23065"
[181] "51154" "100506730" "8574" "54896" "832" "440574"
[187] "644068" "255104" "23252" "55450" "79594" "163933"
[193] "65018" "100861548" "1650" "57576" "400745" "50809"
[199] "8672" "1889" "84224" "249" "5909" "84196"
[205] "401944" "3339" "29092" "998" "54361" "9923"
[211] "2048" "23028" "7798" "100996511" "10236" "80818"
[217] "148898" "6920" "55616" "1870" "3399" "6135"
[223] "6924" "100506963" "57095" "11313" "2582" "3155"
[229] "2517" "55629" "10772" "163702" "57822" "90529"
[235] "57185" "11123" "100506985" "10250" "25932" "25949"
[241] "57035" "23585" "55219" "26119" "57134" "57190"
[247] "56181" "646471" "79000" "164091" "3925" "5051"
[253] "2134" "7780" "84676" "149420" "79927" "51042"
[259] "10256" "64793" "83442" "91544" "79947" "3151"
[265] "6195" "101928728" "8289" "55650" "84243" "54707"
[271] "63906" "10726" "388610" "115572" "6548" "23038"
[277] "84065" "644961" "84958" "9064" "2827" "10163"
[283] "27245" "2537" "199870" "23673" "5511" "6118"
[289] "27293" "55113" "2140" "22826" "93974" "83667"
[295] "54797" "65979" "1104" "8420" "6080" "54952"
[301] "85028" "677838" "6883" "10691" "51441" "2035"
[307] "399474" "6429" "51102" "10076" "9672" "9698"
[313] "79570" "9410" "51538" "2170" "347735" "64129"
[319] "553115" "1307" "576" "90853" "8073" "10657"
[325] "55116" "23633" "200081" "79140" "56063" "55721"
[331] "8668" "339483" "3065" "65108" "100128071" "81629"
[337] "55108" "728116" "653121" "339487" "5928" "81493"
[343] "57648" "8565" "64766" "252995" "113452" "127544"
[349] "204" "113451" "55223" "149076" "1912" "7579"
[355] "114784" "113444" "653160" "100506144" "9204" "79830"
[361] "6421" "9202" "79932" "23154" "5690" "127703"
[367] "63967" "192670" "26523" "192669" "54936" "27095"
[373] "55700" "9967" "79729" "55194" "83931" "84967"
[379] "127700" "64960" "728431" "80149" "64769" "79753"
[385] "7802" "29889" "54955" "55143" "149175" "79693"
[391] "127687" "4520" "3633" "10946" "2275" "51118"
[397] "5453" "64121" "79647" "4725" "23499" "643314"
[403] "353500" "728448" "8761" "26508" "10450" "656"
[409] "54802" "4610" "84879" "10487" "5538" "6018"
[415] "10269" "1298" "64744" "65243" "339559" "64789"
[421] "127396" "9783" "100130557" "4802" "9132" "163732"
[427] "1503" "100507178" "200172" "22955" "100132074" "1907"
[433] "59269" "22887" "284716" "79717" "728621" "10465"
[439] "4904" "64175" "79078" "374969" "114625" "51058"
[445] "6513" "440584" "10969" "149465" "7075" "991"
[451] "64834" "112950" "23334" "5792" "9682" "6487"
[457] "9670" "1802" "533" "8704" "149473" "6536"
[463] "55929" "79033" "55182" "79639" "339541" "11004"
[469] "6202" "266675" "1263" "343521" "149478" "8643"
[475] "8891" "79654" "7389" "57643" "84842" "4595"
[481] "114034" "10420" "126661" "25974" "5052" "10327"
[487] "4678" "149483" "60313" "51249" "3652" "23139"
[493] "8503" "10103" "55624" "541468" "8438" "10489"
[499] "7388" "387338" "2166" "8569" "148932" "64756"
[505] "9813" "6491" "51727" "388630" "54558" "79656"
[511] "11124" "1031" "26994" "22996" "2060" "114883"
[517] "4898" "5865" "51060" "91408" "9372" "200014"
[523] "4998" "84950" "23318" "2882" "65260" "79699"
[529] "55268" "6342" "127435" "1376" "54987" "4116"
[535] "7804" "55706" "54432" "51668" "115353" "9528"
[541] "127428" "606495" "51253" "23648" "619518" "26027"
[547] "338094" "7268" "25973" "1718" "23358" "100507634"
[553] "8613" "5563" "1600" "115209" "114803" "3725"
[559] "55277" "51361" "1573" "4774" "83941" "10207"
[565] "163782" "7398" "85440" "84938" "29929" "23421"
[571] "84455" "5236" "4919" "57685" "55225" "3716"
[577] "205" "9829" "54741" "3953" "5142" "84251"
[583] "79819" "57708" "23169" "26135" "1647" "55970"
[589] "100289178" "9077" "79971" "55635" "57554" "55631"
[595] "9295" "81573" "11147" "1491" "5733" "100132618"
[601] "9406" "406962" "257194" "100526835" "8790" "51086"
[607] "1429" "127253" "204962" "34" "1744" "5876"
[613] "256435" "81849" "10026" "26009" "23032" "374986"
[619] "374987" "91624" "8880" "11080" "54810" "64123"
[625] "23266" "79739" "5567" "80135" "2787" "100505741"
[631] "1486" "117178" "23566" "55283" "126820" "84144"
[637] "148423" "8915" "23576" "3491" "54680" "57489"
[643] "105378828" "51100" "9403" "9653" "8543" "5586"
[649] "2959" "56267" "494115" "2635" "2634" "23507"
[655] "84230" "400761" "55144" "284695" "84146" "164045"
[661] "8317" "7049" "253152" "646817" "23285" "11146"
[667] "79871" "7813" "6125" "388650" "22823" "50999"
[673] "343099" "100131564" "1810" "54874" "8412" "30836"
[679] "2730" "9411" "5825" "2152" "126969" "1266"
[685] "105378862" "729970" "199857" "148534" "100527978" "25950"
[691] "58155" "1806" "51375" "391059" "178" "23443"
[697] "64645" "163786" "54482" "127495" "1629" "100506007"
[703] "8634" "8556" "7412" "2135" "148867" "51611"
[709] "102606465" "101928370" "1901" "1301" "55599" "280"
[715] "55170" "29957" "284611" "113802" "55119" "6814"
[721] "29899" "23155" "22911" "6884" "56900" "57535"
[727] "6301" "1952" "84722" "343263" "6272" "5686"
[733] "284612" "127002" "284613" "57463" "83873" "2773"
[739] "2780" "271" "2948" "2946" "2947" "1435"
[745] "10768" "85369" "257" "388662" "3749" "64783"
[751] "9122" "10542" "3737" "55791" "128338" "10390"
[757] "128344" "5016" "79084" "515" "128346" "100129269"
[763] "5906" "55924" "100506343" "11218" "3752" "55917"
[769] "7482" "54879" "829" "4343" "389" "333926"
[775] "6566" "100506392" "9860" "260425" "10745" "54665"
[781] "10717" "64858" "204851" "56944" "51592" "10286"
[787] "163259" "4893" "7812" "80143" "10100" "4803"
[793] "81839" "845" "55356" "476" "84852" "965"
[799] "3321" "5738" "9398" "8458" "80263" "10905"
[805] "54855" "54834" "10885" "10352" "101929147" "90874"
[811] "26227" "4853" "9554" "728841" "100996724" "101929796"
[817] "101060684" "728855" "653820" "653464" "554282" "100996712"
[823] "284565" "647135" "728833" "103091866" "100288142" "101060254"
[829] "101929780" "653519" "11126" "27246" "10623" "200035"
[835] "10401" "8515" "8799" "9939" "128077" "84265"
[841] "284615" "10628" "148738" "100132406" "388677" "170688"
[847] "149013" "728989" "5565" "171423" "2330" "9557"
[853] "607" "51205" "2702" "51463" "200030" "100132057"
[859] "388685" "25832" "100996763" "440672" "9659" "400818"
[865] "653513" "100996761" "101060226" "653598" "57234" "440689"
[871] "2209" "8370" "105371405" "126961" "8337" "337873"
[877] "723790" "554313" "8349" "51027" "9900" "10262"
[883] "10903" "56957" "11311" "51177" "81611" "23632"
[889] "51107" "79630" "148523" "54460" "9129" "23248"
[895] "80222" "1893" "54507" "4170" "2029" "55204"
[901] "1513" "405" "9869" "29956" "8416" "55793"
[907] "58497" "54964" "56882" "10962" "126626" "10500"
[913] "388695" "79005" "6944" "8394" "5710" "57592"
[919] "5298" "5993" "8991" "5692" "23126" "57530"
[925] "7286" "81609" "65005" "11022" "117145" "6281"
[931] "6282" "6277" "6275" "140576" "6284" "26097"
[937] "23557" "3608" "65123" "11000" "57459" "9909"
[943] "200186" "27173" "148327" "10899" "5872" "6232"
[949] "7170" "25912" "9898" "10456" "89872" "57198"
[955] "3570" "126669" "55585" "103" "3782" "10654"
[961] "57326" "90780" "6464" "1163" "80308" "51043"
[967] "8751" "1945" "1944" "1942" "55974" "54344"
[973] "200185" "80128" "4582" "7059" "4580" "2630"
[979] "2629" "10712" "10067" "1196" "57657" "2224"
[985] "23623" "55870" "645676" "55154" "55249" "7818"
[991] "100129405" "54856" "23208" "6016" "22889" "9181"
[997] "6746" "56893" "28956" "92312" "4000" "64218"
[1003] "9673" "11243" "79957" "23381" "84283" "112770"
[1009] "7203" "4209" "128239" "128240" "54865" "10763"
[1015] "1382" "81875" "51093" "79590" "3068" "5546"
[1021] "375033" "9826" "2117" "55243" "912" "3428"
[1027] "54935" "8407" "57549" "93183" "93185" "477"
[1033] "844" "8682" "50717" "5824" "1314" "474338"
[1039] "23385" "57216" "55600" "50848" "100131187" "7391"
[1045] "257106" "126823" "5202" "4817" "9191" "51506"
[1051] "27005" "5498" "8703" "4720" "84134" "6391"
[1057] "257177" "3310" "11266" "22926" "25903" "9722"
[1063] "400793" "127933" "6675" "4921" "51478" "5999"
[1069] "8490" "83540" "5087" "6258" "4259" "223"
[1075] "54499" "7371" "57645" "117143" "387597" "92235"
[1081] "5451" "8804" "92241" "9019" "25874" "55827"
[1087] "23432" "261726" "375035" "84832" "9095" "101928596"
[1093] "481" "29922" "8548" "57821" "10560" "2153"
[1099] "6403" "92342" "55732" "57147" "22920" "149281"
[1105] "92344" "5396" "2329" "23215" "8674" "51603"
[1111] "26052" "92346" "5279" "51430" "7292" "100506023"
[1117] "9588" "339416" "27252" "91687" "55157" "100506046"
[1123] "60674" "26802" "26774" "26770" "84614" "149041"
[1129] "9910" "27101" "63931" "9674" "64326" "60676"
[1135] "460" "57795" "100302401" "9462" "400798" "55103"
[1141] "9917" "64222" "27" "6646" "163590" "26092"
[1147] "9857" "5768" "200058" "89884" "100527964" "84320"
[1153] "148756" "9213" "57710" "10228" "51278" "2752"
[1159] "6041" "6004" "80896" "1660" "3915" "3918"
[1165] "23057" "284649" "9887" "10092" "23179" "23127"
[1171] "116461" "81563" "80267" "116496" "6045" "81627"
[1177] "54823" "10625" "83872" "7175" "54953" "5743"
[1183] "339479" "101929120" "100616345" "5997" "51377" "6738"
[1189] "51022" "79577" "8707" "343450" "259266" "360023"
[1195] "163486" "388722" "140609" "2494" "23528" "9928"
[1201] "83479" "23271" "23046" "779" "252839" "7139"
[1207] "3898" "7135" "23612" "1465" "89796" "55705"
[1213] "149345" "25802" "10440" "6051" "1999" "127829"
[1219] "29089" "4660" "127833" "10765" "100506696" "5877"
[1225] "59349" "51094" "51706" "92703" "8497" "134"
[1231] "7832" "493" "84719" "9877" "100381270" "6635"
[1237] "9580" "55224" "22874" "84919" "5287" "4194"
[1243] "10446" "23114" "388730" "5929" "25778" "9911"
[1249] "81788" "55220" "5129" "148808" "2005" "85414"
[1255] "64710" "8934" "254428" "284581" "115019" "103021295"
[1261] "338382" "729533" "23380" "9641" "83593" "1939"
[1267] "8444" "9261" "5208" "55432" "1604" "4179"
[1273] "5362" "3914" "50486" "148304" "3664" "27042"
[1279] "255928" "574036" "56256" "55733" "55758" "7188"
[1285] "84791" "7779" "4751" "9926" "25896" "51514"
[1291] "100131205" "5525" "55248" "29937" "467" "25936"
[1297] "128387" "642946" "28982" "79805" "90806" "26750"
[1303] "100505832" "5629" "56950" "5784" "1063" "51133"
[1309] "2104" "55105" "51018" "7042" "127018" "2058"
[1315] "10380" "55699" "25782" "6791" "4139" "79762"
[1321] "54996" "64757" "3142" "11221" "9015" "375056"
[1327] "64853" "148362" "84976" "55061" "824" "7159"
[1333] "105369195" "23219" "8560" "4931" "29097" "80232"
[1339] "149111" "127602" "3930" "55740" "6726" "2052"
[1345] "9725" "10637" "29920" "7044" "163859" "3020"
[1351] "64746" "83881" "286826" "142" "375057" "3707"
[1357] "5664" "56997" "8476" "339500" "100130093" "65094"
[1363] "116841" "7483" "375" "79169" "128308" "2987"
[1369] "200205" "84033" "101927401" "81559" "51127" "92815"
[1375] "149603" "58480" "5867" "126731" "58" "55746"
[1381] "23456" "27097" "9816" "2590" "79605" "22796"
[1387] "183" "84886" "101927604" "79573" "64801" "375061"
[1393] "440730" "128061" "8443" "149371" "83932" "54583"
[1399] "7257" "27184" "57568" "54627" "84284" "80003"
[1405] "84451" "3775" "388753" "6894" "359948" "9804"
[1411] "23029" "51742" "9453" "6905" "148789" "1130"
[1417] "4811" "7107" "56605" "128178" "3964" "55127"
[1423] "88" "4548" "6262" "1131" "56776" "2271"
[1429] "23596" "1122" "9156" "200150" "731275" "9859"
[1435] "10806" "10000" "10472" "159" "51029" "116228"
[1441] "284702" "3192" "101928068" "84288" "55083" "64754"
[1447] "64216" "163882" "51097" "100130097" "25909" "93474"
[1453] "79862" "388759" "7678" "84838" "25893" "343171"
[1459] "80851" "79894" "55657" "267002" "10771" "22982"
[1465] "23185" "23560" "91734" "3422" "22884" "101927824"
[1471] "5214" "10531" "100507034" "1316" "100652988" "1645"
[1477] "10276" "79754" "54906" "2665" "54522" "84893"
[1483] "84991" "5209" "5588" "439949" "57713" "80760"
[1489] "22944" "509" "83860" "10659" "414196" "9712"
[1495] "79746" "254427" "219731" "26019" "55526" "55176"
[1501] "11164" "8872" "57118" "83643" "10133" "55388"
[1507] "5264" "22929" "222389" "8559" "55691" "83641"
[1513] "441549" "51182" "79723" "64421" "414149" "221060"
[1519] "10557" "9397" "221061" "8516" "80013" "9317"
[1525] "389941" "6251" "8029" "1787" "100507347" "7431"
[1531] "338596" "9200" "102723166" "8027" "783" "221078"
[1537] "221079" "84898" "10529" "399726" "387640" "8028"
[1543] "64215" "23412" "648" "100499489" "5305" "22921"
[1549] "256815" "220213" "56243" "57584" "56952" "79896"
[1555] "57512" "23590" "10006" "22852" "10730" "84930"
[1561] "91452" "22931" "143098" "220906" "51322" "25805"
[1567] "102724316" "6840" "57608" "55149" "1326" "645954"
[1573] "220929" "220930" "6935" "94134" "3799" "80314"
[1579] "79741" "3688" "8829" "56288" "8453" "1390"
[1585] "219771" "8325" "57209" "219749" "7581" "7587"
[1591] "399744" "441666" "100129482" "7582" "9790" "5979"
[1597] "55454" "221002" "3185" "8187" "220992" "7580"
[1603] "414208" "6387" "83937" "11067" "220979" "7570"
[1609] "220972" "93550" "253725" "119016" "728407" "100287932"
[1615] "8031" "653268" "728113" "643650" "5540" "9721"
[1621] "83849" "414241" "399761" "2662" "2658" "118738"
[1627] "653145" "642517" "642826" "439965" "102724488" "100996758"
[1633] "414224" "143162" "5599" "58504" "196740" "118461"
[1639] "2074" "55753" "8505" "100652748" "414189" "387680"
[1645] "56624" "259230" "104355295" "653308" "5592" "23283"
[1651] "22943" "11130" "253430" "55847" "7321" "7019"
[1657] "80114" "220965" "220963" "8030" "288" "983"
[1663] "9886" "84159" "219790" "84890" "1959" "29982"
[1669] "221037" "84989" "221035" "29119" "347731" "23411"
[1675] "26091" "84665" "64081" "3189" "55680" "1763"
[1681] "8034" "80312" "55749" "219736" "79009" "9188"
[1687] "26128" "9559" "6832" "80201" "3098" "6865"
[1693] "23555" "219738" "1305" "55506" "84883" "219743"
[1699] "56681" "5464" "55222" "1979" "27143" "140766"
[1705] "8879" "5092" "219699" "728978" "55315" "64115"
[1711] "5660" "9469" "9806" "51008" "119504" "54541"
[1717] "54788" "10367" "90550" "5033" "25961" "11319"
[1723] "317662" "23234" "414245" "51021" "118491" "310"
[1729] "118490" "5532" "159195" "58529" "79933" "729092"
[1735] "729096" "2749" "9632" "170384" "118487" "23053"
[1741] "100507331" "8509" "818" "5328" "7414" "26985"
[1747] "132" "23522" "51207" "142891" "7417" "118881"
[1753] "84858" "100131213" "3778" "9231" "100128292" "11128"
[1759] "6229" "57178" "10105" "219654" "143244" "650623"
[1765] "101060691" "729262" "642361" "80195" "311" "84293"
[1771] "81619" "10718" "27069" "92211" "54462" "2894"
[1777] "23063" "11155" "657" "79812" "6623" "2746"
[1783] "54537" "728118" "728190" "439994" "728130" "9562"
[1789] "9060" "84896" "142913" "100144748" "5728" "57559"
[1795] "100132116" "59" "355" "3988" "3437" "3434"
[1801] "24138" "387700" "53354" "9585" "3363" "10556"
[1807] "27063" "119369" "84333" "143279" "5507" "80351"
[1813] "143282" "9044" "22849" "54708" "3416" "3832"
[1819] "54536" "26509" "55165" "118924" "159371" "266971"
[1825] "51196" "64318" "23232" "3070" "9124" "10580"
[1831] "5832" "26123" "953" "54619" "9849" "7093"
[1837] "56889" "118788" "728658" "102723665" "84458" "26148"
[1843] "84986" "10023" "23401" "23223" "644215" "5223"
[1849] "51013" "84287" "64210" "80019" "118812" "55361"
[1855] "60370" "83742" "118813" "6425" "401647" "55118"
[1861] "27291" "84171" "84795" "3257" "26507" "2805"
[1867] "81894" "57089" "1355" "51076" "23268" "10613"
[1873] "1147" "55280" "282991" "6319" "90271" "25956"
[1879] "4714" "55662" "55719" "57715" "84545" "56652"
[1885] "84445" "81855" "81621" "8945" "27343" "25911"
[1891] "6468" "10360" "10724" "30819" "79591" "79803"
[1897] "8861" "23082" "9221" "8729" "4791" "5662"
[1903] "79176" "79004" "100505761" "79847" "10121" "51684"
[1909] "81603" "403" "118980" "54838" "119032" "54805"
[1915] "22978" "729020" "9118" "84108" "6877" "84833"
[1921] "22984" "51063" "9148" "9644" "79991" "9748"
[1927] "119392" "9446" "119391" "85450" "159686" "22986"
[1933] "114815" "7511" "120" "4601" "10285" "1847"
[1939] "9126" "282996" "282997" "27250" "92482" "8036"
[1945] "150" "57678" "64429" "143187" "6934" "4892"
[1951] "840" "9937" "374354" "153" "55088" "84632"
[1957] "3983" "57700" "142940" "26033" "2674" "259217"
[1963] "57698" "6571" "118987" "22841" "63877" "143384"
[1969] "340719" "8661" "404636" "119559" "10935" "2869"
[1975] "6001" "7073" "9531" "22876" "79892" "11196"
[1981] "196051" "55717" "2263" "11101" "54780" "10579"
[1987] "59338" "5654" "80007" "118672" "64376" "36"
[1993] "9184" "119587" "51363" "4942" "64077" "9679"
[1999] "399818" "23172" "54764" "1488" "26098" "7390"
[2005] "56647" "55760" "92565" "8038" "1793" "642938"
[2011] "5791" "4288" "4255" "387723" "119437" "10539"
[2017] "399827" "55844" "664" "282973" "10570" "282974"
[2023] "80313" "170394" "3632" "54777" "85442" "101"
[2029] "10844" "118472" "282969" "1892" "196743" "92170"
[2035] "503542" "100133161" "51272" "60626" "23410" "5719"
[2041] "80162" "10581" "8519" "10410" "338707" "59307"
[2047] "81490" "6050" "115399" "3265" "256329" "8045"
[2053] "100506211" "57661" "3665" "10522" "283232" "64787"
[2059] "6888" "347862" "171391" "105369190" "51286" "79751"
[2065] "101927423" "55367" "6181" "57104" "283229" "977"
[2071] "5441" "7106" "66005" "161" "54472" "9024"
[2077] "81532" "1850" "338651" "402778" "1509" "6150"
[2083] "283120" "3481" "51214" "29125" "10077" "975"
[2089] "10078" "3784" "10984" "1028" "5002" "7262"
[2095] "4676" "833" "114879" "7748" "650368" "116969"
[2101] "4928" "27315" "391" "6786" "6240" "6737"
[2107] "55128" "117854" "85363" "10346" "84067" "112464"
[2113] "6609" "322" "10612" "23647" "26515" "144132"
[2119] "23378" "3611" "6881" "1200" "8642" "63875"
[2125] "7762" "7761" "143425" "8495" "51700" "8665"
[2131] "7275" "79608" "65975" "9866" "6157" "6764"
[2137] "56672" "56674" "493900" "56675" "57758" "283102"
[2143] "23258" "440026" "10527" "644656" "7702" "7465"
[2149] "23075" "440028" "283104" "81846" "105376544" "133"
[2155] "272" "100463486" "50862" "10335" "9646" "1982"
[2161] "58486" "729013" "374378" "283106" "55031" "27122"
[2167] "9645" "84953" "55742" "7003" "406" "84280"
[2173] "84188" "10418" "22800" "1315" "5682" "5140"
[2179] "120227" "55553" "10944" "144100" "6207" "5286"
[2185] "4925" "374383" "3767" "26297" "113174" "11234"
[2191] "2965" "3939" "7251" "55293" "144108" "144110"
[2197] "54503" "8048" "79733" "89797" "100126784" "100874014"
[2203] "10553" "10196" "203859" "2188" "2620" "258010"
[2209] "159963" "387758" "91057" "55366" "55327" "497258"
[2215] "627" "81930" "196074" "3739" "120534" "744"
[2221] "120526" "196294" "26610" "5954" "7490" "10480"
[2227] "79832" "91614" "55346" "283267" "1479" "10114"
[2233] "25758" "966" "26273" "4005" "4076" "55226"
[2239] "25841" "847" "51074" "8050" "960" "6506"
[2245] "24147" "54765" "143458" "29099" "79899" "7189"
[2251] "5896" "119710" "8539" "55761" "51144" "221120"
[2257] "387763" "84680" "2132" "3732" "90139" "9537"
[2263] "56981" "57586" "8534" "55343" "1408" "9479"
[2269] "9409" "51317" "90993" "8525" "4192" "1132"
[2275] "55626" "283254" "9776" "392" "79797" "9793"
[2281] "4038" "79096" "84364" "29763" "1643" "53"
[2287] "10062" "8567" "4607" "91252" "5702" "10658"
[2293] "114971" "55709" "4722" "23788" "23360" "23279"
[2299] "5795" "85456" "6749" "29015" "8501" "26519"
[2305] "9246" "710" "219539" "10978" "25921" "219541"
[2311] "51075" "280636" "1500" "9404" "80829" "374393"
[2317] "63901" "23220" "5007" "219988" "6809" "54948"
[2323] "79080" "27339" "79073" "54972" "55048" "220001"
[2329] "1642" "26007" "220002" "51524" "51259" "79869"
[2335] "54949" "9066" "113157" "747" "745" "746"
[2341] "2237" "3992" "9415" "3995" "5866" "7439"
[2347] "2495" "3619" "80150" "79026" "1937" "64852"
[2353] "9219" "256364" "6094" "26229" "23193" "80789"
[2359] "102288414" "79081" "751071" "790955" "51035" "26580"
[2365] "221092" "283237" "79842" "5436" "10629" "374395"
[2371] "79064" "10482" "6811" "54663" "23642" "9304"
[2377] "9298" "9303" "6520" "11145" "25923" "10313"
[2383] "65998" "144097" "2011" "283248" "79829" "1351"
[2389] "55611" "28992" "10963" "83706" "83707" "84304"
[2395] "3338" "7423" "2286" "26472" "5331" "56834"
[2401] "572" "2101" "51504" "25824" "283234" "8986"
[2407] "10235" "5837" "7536" "5871" "4221" "55561"
[2413] "10938" "23130" "5526" "402" "29901" "113130"
[2419] "7542" "738" "7108" "741" "2197" "740"
[2425] "84447" "728975" "823" "23649" "10435" "5977"
[2431] "283130" "83786" "283131" "101927789" "378938" "57410"
[2437] "4054" "254100" "10534" "23625" "254102" "4296"
[2443] "399909" "6494" "5970" "10524" "84153" "91056"
[2449] "254122" "1072" "80198" "30008" "9158" "11007"
[2455] "8061" "83638" "10589" "9092" "84285" "8815"
[2461] "89792" "10992" "55690" "64837" "81876" "254263"
[2467] "10897" "256472" "57124" "9610" "25855" "11041"
[2473] "3177" "65003" "246330" "10072" "582" "254359"
[2479] "89" "8722" "55231" "9973" "10432" "5936"
[2485] "83759" "6712" "79703" "9986" "5091" "78999"
[2491] "91683" "29984" "22992" "156" "338692" "54961"
[2497] "57804" "23529" "5883" "5499" "374403" "57571"
[2503] "6199" "5790" "57175" "80194" "9049" "9600"
[2509] "10263" "2950" "4723" "390213" "254552" "347853"
[2515] "645332" "81622" "221" "4728" "10312" "1119"
[2521] "51111" "53838" "4041" "55291" "51083" "9633"
[2527] "1374" "219927" "3508" "219931" "595" "220064"
[2533] "55107" "8772" "8500" "2017" "1717" "55191"
[2539] "55199" "100133315" "55298" "10068" "4926" "100128494"
[2545] "220074" "55004" "25906" "2348" "3636" "81570"
[2551] "5138" "116985" "10809" "89849" "9873" "5029"
[2557] "9828" "84957" "23201" "58473" "5870" "51642"
[2563] "51287" "80227" "7351" "26005" "51400" "283208"
[2569] "283209" "387787" "100287896" "10714" "254225" "143570"
[2575] "9789" "10825" "100507050" "408" "6188" "283212"
[2581] "81544" "871" "4135" "84649" "7405" "7481"
[2587] "5612" "100506127" "56946" "2615" "25987" "55331"
[2593] "726" "5058" "282679" "1207" "51773" "28971"
[2599] "92105" "4718" "79053" "283219" "57558" "9846"
[2605] "79731" "26011" "5547" "220042" "27314" "100506233"
[2611] "51585" "338699" "60492" "1740" "55863" "84233"
[2617] "58487" "54843" "8301" "8726" "51501" "10873"
[2623] "11098" "8322" "65084" "1075" "50507" "10003"
[2629] "26973" "120114" "120103" "56935" "85459" "684959"
[2635] "692063" "654320" "692072" "677805" "677822" "79101"
[2641] "28970" "9440" "24145" "4361" "54851" "2526"
[2647] "143689" "154810" "51503" "55693" "10929" "23052"
[2653] "143686" "100129203" "143684" "9702" "8898" "84441"
[2659] "79780" "8690" "53942" "143872" "83935" "5241"
[2665] "57562" "10413" "330" "329" "114908" "4319"
[2671] "4312" "84259" "79659" "80310" "837" "84437"
[2677] "143879" "60496" "2977" "143884" "91801" "55531"
[2683] "54733" "8065" "38" "4863" "472" "143888"
[2689] "23086" "1662" "85463" "5962" "2230" "57569"
[2695] "143903" "23235" "5519" "79796" "91893" "64776"
[2701] "1410" "3316" "85458" "1737" "120379" "55216"
[2707] "26521" "6392" "83875" "5805" "101928847" "4684"
[2713] "100288346" "54970" "1813" "9183" "57646" "4837"
[2719] "54494" "10179" "25996" "23705" "84811" "8882"
[2725] "335" "23387" "5049" "51092" "100652768" "6876"
[2731] "9159" "257160" "23621" "100379571" "22897" "53826"
[2737] "6330" "6327" "196264" "9354" "10632" "4297"
[2743] "84866" "56912" "372" "23187" "1656" "283149"
[2749] "338657" "6230" "51399" "2542" "10525" "55823"
[2755] "3145" "3014" "1798" "9854" "25988" "64137"
[2761] "79671" "867" "4162" "79102" "9099" "7070"
[2767] "5818" "220323" "219902" "23365" "2900" "219899"
[2773] "7007" "6309" "6653" "399959" "84959" "79864"
[2779] "3312" "57476" "55800" "7753" "4013" "84897"
[2785] "54414" "53340" "79684" "100507283" "219854" "63876"
[2791] "9638" "9538" "3703" "1111" "219844" "83480"
[2797] "50937" "84881" "79607" "6734" "55572" "114609"
[2803] "28960" "6484" "2113" "2313" "3762" "219833"
[2809] "9743" "4798" "56980" "334" "6768" "29068"
[2815] "11095" "170689" "103611081" "399979" "50863" "4978"
[2821] "283174" "22997" "83700" "23310" "112936" "29087"
[2827] "27034" "89944" "27087" "100288778" "6539" "6540"
[2833] "5927" "84318" "283358" "4815" "100049716" "65125"
[2839] "5893" "23085" "81029" "144699" "79602" "196513"
[2845] "775" "2288" "55846" "83714" "2305" "83695"
[2851] "7289" "7004" "10867" "57097" "103752584" "894"
[2857] "57103" "57102" "10635" "8798" "10566" "4704"
[2863] "3742" "3736" "3741" "4908" "928" "7132"
[2869] "4055" "678655" "55080" "6843" "51258" "9918"
[2875] "2597" "25900" "4839" "1108" "57121" "51147"
[2881] "171017" "196500" "50813" "8079" "5763" "27239"
[2887] "10536" "2784" "83461" "8078" "7167" "84727"
[2893] "171220" "10233" "2026" "1822" "113246" "5777"
[2899] "11331" "10436" "10162" "716" "715" "51279"
[2905] "9746" "5830" "144195" "6515" "55810" "25977"
[2911] "440078" "8076" "57494" "1911" "4074" "10219"
[2917] "144571" "2" "100499405" "642846" "440081" "374443"
[2923] "29121" "387836" "4973" "23710" "10748" "55110"
[2929] "8531" "100129361" "2120" "100506248" "4040" "54682"
[2935] "503693" "118426" "80824" "1389" "1027" "81575"
[2941] "51202" "387841" "9052" "50865" "93164" "57613"
[2947] "83445" "2012" "2904" "55729" "79887" "105369669"
[2953] "55766" "144608" "51729" "397" "85004" "2059"
[2959] "11171" "51071" "4257" "55885" "728622" "54477"
[2965] "121536" "5139" "79912" "5965" "51026" "80763"
[2971] "2998" "3945" "3764" "10060" "55907" "9847"
[2977] "55500" "6660" "586" "144363" "3845" "100506451"
[2983] "11228" "79365" "8082" "3709" "55726" "26127"
[2989] "51768" "9412" "23012" "56938" "8496" "387849"
[2995] "60488" "57542" "5744" "55297" "55711" "100506606"
[3001] "51290" "83857" "10526" "65981" "441631" "1663"
[3007] "58516" "160518" "100874249" "254013" "196394" "440093"
[3013] "55196" "636" "121512" "10059" "51067" "5318"
[3019] "84920" "144245" "144402" "55605" "114134" "120892"
[3025] "283463" "1272" "283464" "10138" "85437" "51535"
[3031] "144165" "83448" "51135" "5756" "84216" "4753"
[3037] "51054" "196527" "400027" "196528" "9169" "81539"
[3043] "54407" "100288798" "55089" "347902" "79657" "10411"
[3049] "55652" "51564" "7421" "79022" "1280" "29843"
[3055] "5213" "140461" "121274" "54934" "904" "112"
[3061] "784" "9416" "27289" "85478" "51303" "377"
[3067] "23109" "5571" "8085" "55716" "10376" "7846"
[3073] "84790" "10024" "79962" "65244" "10445" "84070"
[3079] "25766" "91010" "7009" "57701" "100286844" "144233"
[3085] "29127" "41" "6602" "2819" "84987" "91012"
[3091] "51474" "113251" "57609" "466" "25840" "4891"
[3097] "25875" "81566" "7024" "5463" "9802" "11226"
[3103] "9498" "6334" "401720" "91" "160622" "3164"
[3109] "60673" "144501" "3855" "3856" "3875" "1975"
[3115] "283335" "23371" "84926" "3489" "51380" "283337"
[3121] "5916" "84975" "9700" "5204" "60314" "8086"
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[14089] "3188" "100131755" "51309" "54470" "51566" "9823"
[14095] "84460" "11013" "64860" "100528062" "9737" "80823"
[14101] "100287765" "55859" "56271" "90843" "84707" "56849"
[14107] "51186" "27018" "79921" "85012" "9338" "9643"
[14113] "51209" "286527" "767811" "401612" "644353" "286437"
[14119] "139231" "203447" "139221" "55086" "79589" "54885"
[14125] "79710" "55285" "54830" "5631" "1831" "11043"
[14131] "5716" "115201" "1288" "1287" "55916" "2182"
[14137] "105373311" "84187" "9949" "91851" "79868" "154796"
[14143] "57631" "101927352" "5358" "90293" "54521" "139818"
[14149] "3597" "170261" "10857" "203427" "100303728" "292"
[14155] "63932" "7319" "55922" "23157" "6170" "65109"
[14161] "7737" "4694" "79576" "10009" "55026" "23439"
[14167] "3920" "8450" "28985" "29071" "2747" "2892"
[14173] "57187" "331" "10735" "10178" "6594" "4952"
[14179] "8862" "51114" "10813" "63035" "2000" "9131"
[14185] "9363" "55609" "9016" "51634" "55855" "10495"
[14191] "3547" "286467" "51765" "57826" "101928578" "55796"
[14197] "90161" "2239" "2719" "347475" "84295" "3251"
[14203] "84848" "159090" "159091" "56180" "644538" "441518"
[14209] "8933" "26071" "644596" "7626" "203523" "399668"
[14215] "203522" "93380" "10479" "2273" "79649" "27336"
[14221] "9459" "27316" "101928747" "158696" "2258" "100129662"
[14227] "4168" "286410" "23641" "139065" "2332" "2334"
[14233] "3423" "100131434" "91966" "84548" "100506164" "541578"
[14239] "100272228" "10046" "4534" "8776" "83692" "3149"
[14245] "9248" "203547" "79057" "2564" "1069" "50814"
[14251] "7739" "29944" "10838" "55559" "633" "92002"
[14257] "139728" "6535" "10134" "215" "5365" "26576"
[14263] "3421" "6748" "57595" "3897" "393" "8260"
[14269] "5973" "3054" "8269" "3654" "4204" "2316"
[14275] "2010" "6134" "26778" "1774" "6901" "158960"
[14281] "537" "2664" "9130" "55558" "8270" "8266"
[14287] "8273" "60343" "2539" "8517" "139716" "1736"
[14293] "4354" "2157" "8263" "65991" "100272147" "4515"
[14299] "79184" "7411" "116442" "101927830" "474383" "474384"
[14305] "55217" "10251" "6845" "105379416" "6192" "7544"
[14311] "5616" "64595" "8287" "8653" "7404" "22829"
[14317] "246126" "8284" "9086" comparison_DEGs <- list(
DOX_RM = as.character(DOX_RM_DEGs$Entrez_ID),
DNR_RM = as.character(DNR_RM_DEGs$Entrez_ID),
EPI_RM = as.character(EPI_RM_DEGs$Entrez_ID),
MTX_RM = as.character(MTX_RM_DEGs$Entrez_ID),
CX_SP = as.character(CX_SP_DEGs$Entrez_ID),
DOX_SP = as.character(DOX_SP_DEGs$Entrez_ID)
)
#fisher's exact test per treatment
fisher_test_overlap <- function(acute, chronic, degs) {
a <- sum(acute %in% degs)
b <- length(acute) - a
c <- sum(chronic %in% degs)
d <- length(chronic) - c
contingency <- matrix(c(a, b, c, d), nrow = 2, byrow = TRUE)
fisher_res <- fisher.test(contingency)
list(
contingency = contingency,
p_value = fisher_res$p.value,
significant = case_when(
fisher_res$p.value < 0.001 ~ "***",
fisher_res$p.value < 0.01 ~ "**",
fisher_res$p.value < 0.05 ~ "*",
TRUE ~ ""
)
)
}
# Prepare summary table for plotting
plot_summary <- map_dfr(names(comparison_DEGs), function(treat) {
degs <- comparison_DEGs[[treat]]
# Fisher test
fisher_res <- fisher_test_overlap(acute_set, chronic_set, degs)
# Counts for each motif
acute_overlap <- sum(acute_set %in% degs)
acute_nonoverlap <- length(acute_set) - acute_overlap
chronic_overlap <- sum(chronic_set %in% degs)
chronic_nonoverlap <- length(chronic_set) - chronic_overlap
tibble(
Treatment = treat,
Motif = rep(c("Acute", "Chronic"), each = 2),
Category = rep(c("DEG", "nonDEG"), 2),
Count = c(acute_overlap, acute_nonoverlap, chronic_overlap, chronic_nonoverlap),
Fraction = c(acute_overlap, acute_nonoverlap, chronic_overlap, chronic_nonoverlap) /
c(length(acute_set), length(acute_set), length(chronic_set), length(chronic_set)),
Significant = rep(c(fisher_res$significant, ""), 2) #only on DEG bars
)
})
#ensure correct order
plot_summary$Category <- factor(plot_summary$Category, levels = c("DEG", "nonDEG"))
#position stars at y = 1
label_positions <- plot_summary %>%
filter(Category == "DEG") %>%
mutate(y_pos = 1)
#plot
top2i_barplot_motifs_supp7 <- ggplot(plot_summary, aes(x = Treatment, y = Fraction, fill = Category)) +
geom_bar(stat = "identity", position = "stack") +
geom_text(data = label_positions %>% filter(Significant != ""),
aes(x = Treatment, y = y_pos, label = Significant),
inherit.aes = FALSE, size = 6, fontface = "bold", color = "black") +
geom_text(aes(label = paste0(round(Fraction*100,1), "")),
position = position_stack(vjust = 0.5), size = 5, color = "white") +
scale_y_continuous(limits = c(0, 1)) +
scale_fill_manual(values = c("DEG" = "blue", "nonDEG" = "red")) +
facet_wrap(~Motif) +
labs(
title = "Percentage of TOP2i DEGs (Matthews et al., Paul et al.) in Acute and Chronic response clusters",
x = "TOP2i Treatment",
y = "Overlap Percentage (%)",
fill = ""
) +
theme_custom()
print(top2i_barplot_motifs_supp7)
| Version | Author | Date |
|---|---|---|
| 7f22593 | emmapfort | 2026-01-05 |
# save_plot(
# plot = top2i_barplot_motifs_supp7,
# filename = "Supp7_TOP2i_Barplots_EMP",
# folder = output_folder,
# height = 5,
# width = 8.5
# )
#print percentages and p-values
for(treat in names(comparison_DEGs)) {
degs <- comparison_DEGs[[treat]]
acute_overlap <- sum(acute_set %in% degs)
chronic_overlap <- sum(chronic_set %in% degs)
prop_ac <- acute_overlap / length(acute_set)
prop_ch <- chronic_overlap / length(chronic_set)
fisher_res <- fisher_test_overlap(acute_set, chronic_set, degs)
cat(sprintf("%s - Acute: %.2f%% (%d/%d), Chronic: %.2f%% (%d/%d), Fisher p = %.5f\n",
treat, 100*prop_ac, acute_overlap, length(acute_set),
100*prop_ch, chronic_overlap, length(chronic_set),
fisher_res$p_value))
}DOX_RM - Acute: 44.50% (3383/7602), Chronic: 69.66% (349/501), Fisher p = 0.00000
DNR_RM - Acute: 46.97% (3571/7602), Chronic: 68.66% (344/501), Fisher p = 0.00000
EPI_RM - Acute: 40.87% (3107/7602), Chronic: 65.87% (330/501), Fisher p = 0.00000
MTX_RM - Acute: 5.71% (434/7602), Chronic: 56.89% (285/501), Fisher p = 0.00000
CX_SP - Acute: 0.50% (38/7602), Chronic: 40.52% (203/501), Fisher p = 0.00000
DOX_SP - Acute: 64.84% (4929/7602), Chronic: 68.26% (342/501), Fisher p = 0.12188#significance stars are for likelihood of being in the Chronic cluster over the Acute cluster - stars are the same on both sides but only really count for ChronicFigure 4B - Enrichment of TOP2i DEGs in Chronic cluster relative to Acute
#use the workflow above to now plot forest plots of odds ratios for these comparisons
#I am comparing sets of DEGs from other TOP2i against my motifs here
#I want to use my fisher's exact test data
#same inputs as before to preserve them
# final_genes_1_RUV
# final_genes_2_RUV
# acute_set
# chronic_set
#remove DOX and preserve order DNR EPI MTX CX
comparison_DEGs_noDOX <- list(
DNR_RM = as.character(DNR_RM_DEGs$Entrez_ID),
EPI_RM = as.character(EPI_RM_DEGs$Entrez_ID),
MTX_RM = as.character(MTX_RM_DEGs$Entrez_ID),
CX_SP = as.character(CX_SP_DEGs$Entrez_ID)
)
treatment_order <- c("DNR_RM", "EPI_RM", "MTX_RM", "CX_SP")
fisher_test_overlap <- function(acute, chronic, degs) {
a <- sum(acute %in% degs)
b <- length(acute) - a
c <- sum(chronic %in% degs)
d <- length(chronic) - c
contingency <- matrix(c(a, b, c, d), nrow = 2, byrow = TRUE)
fisher_res <- fisher.test(contingency)
tibble(
OR = unname(fisher_res$estimate),
CI_low = fisher_res$conf.int[1],
CI_high = fisher_res$conf.int[2],
p_value = fisher_res$p.value,
Significant = case_when(
fisher_res$p.value < 0.001 ~ "***",
fisher_res$p.value < 0.01 ~ "**",
fisher_res$p.value < 0.05 ~ "*",
TRUE ~ "ns"
)
)
}
#build a summary table for the results of fishers exact for the forest plots
forest_summary <- map_dfr(names(comparison_DEGs_noDOX), function(treat) {
res <- fisher_test_overlap(chronic_set, acute_set, comparison_DEGs_noDOX[[treat]])
res %>% mutate(Treatment = treat)
}) %>%
mutate(Treatment = factor(Treatment, levels = rev(treatment_order)), #define order
Group = ifelse(grepl("_RM$", Treatment), "RM", "SP"))
#create my combined plot
forest_TOP2i <- ggplot(forest_summary,
aes(y = Treatment, x = OR,
xmin = CI_low, xmax = CI_high)) +
geom_point(size = 4) +
geom_errorbarh(height = 0.2, linewidth = 0.5, color = "black") +
geom_text(aes(label = Significant), position = position_nudge(y = 0.1),
color = "black", fontface = "bold", size = 5) +
geom_vline(xintercept = 1, linetype = "dashed", color = "red") +
scale_x_log10(limits = c(0.1, 1000),
breaks = c(0.1, 1, 10, 100, 1000)) +
scale_color_manual(values = c("_" = "grey", "_" = "grey")) + #remove the groups this way even though it's not the best method
labs(
title = "Enrichment of TOP2i DEGs in Chronic response cluster relative to Acute response cluster",
x = "log odds ratio (95% confidence interval)",
y = ""
) +
theme_custom()
print(forest_TOP2i)
| Version | Author | Date |
|---|---|---|
| 7f22593 | emmapfort | 2026-01-05 |
#make a summary table
forest_summary_TOP2i <- forest_summary
View(forest_summary_TOP2i)
sessionInfo()R version 4.4.2 (2024-10-31 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 11 x64 (build 22000)
Matrix products: default
locale:
[1] LC_COLLATE=English_United States.utf8
[2] LC_CTYPE=English_United States.utf8
[3] LC_MONETARY=English_United States.utf8
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.utf8
time zone: America/Chicago
tzcode source: internal
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] readxl_1.4.5 lubridate_1.9.4 forcats_1.0.0 stringr_1.5.1
[5] dplyr_1.1.4 purrr_1.0.4 readr_2.1.5 tidyr_1.3.1
[9] tibble_3.2.1 ggplot2_3.5.2 tidyverse_2.0.0 workflowr_1.7.1
loaded via a namespace (and not attached):
[1] utf8_1.2.5 sass_0.4.10 generics_0.1.4 stringi_1.8.7
[5] hms_1.1.3 digest_0.6.37 magrittr_2.0.3 timechange_0.3.0
[9] evaluate_1.0.3 grid_4.4.2 RColorBrewer_1.1-3 fastmap_1.2.0
[13] cellranger_1.1.0 rprojroot_2.0.4 jsonlite_2.0.0 processx_3.8.6
[17] whisker_0.4.1 ps_1.9.1 promises_1.3.2 httr_1.4.7
[21] scales_1.4.0 jquerylib_0.1.4 cli_3.6.3 rlang_1.1.6
[25] withr_3.0.2 cachem_1.1.0 yaml_2.3.10 tools_4.4.2
[29] tzdb_0.5.0 httpuv_1.6.16 vctrs_0.6.5 R6_2.6.1
[33] lifecycle_1.0.4 git2r_0.36.2 fs_1.6.6 pkgconfig_2.0.3
[37] callr_3.7.6 pillar_1.10.2 bslib_0.9.0 later_1.4.2
[41] gtable_0.3.6 glue_1.8.0 Rcpp_1.0.14 xfun_0.52
[45] tidyselect_1.2.1 rstudioapi_0.17.1 knitr_1.50 farver_2.1.2
[49] htmltools_0.5.8.1 labeling_0.4.3 rmarkdown_2.29 compiler_4.4.2
[53] getPass_0.2-4