Figure 4 Analysis
Last updated: 2026-01-05
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Knit directory: DXR_website/
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| 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.
#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"
[3127] "6667" "269" "54458" "5094" "7786" "6895"
[3133] "11016" "517" "57658" "23583" "23468" "3178"
[3139] "22818" "25946" "3678" "5502" "3679" "2647"
[3145] "967" "10220" "84324" "29095" "85406" "440104"
[3151] "84305" "102724112" "1606" "6490" "1017" "5869"
[3157] "6821" "64375" "6231" "2065" "5036" "6171"
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[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)
# 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)
#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