Peak Calling cutoff (RAD21 and CTCF)

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Last updated: 2025-08-25

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Knit directory: ChIPSeq_project/

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---

📌 CTCF peaks cutoff (before deduplication)

library(tidyverse)

Warning: package 'tidyverse' was built under R version 4.3.2

Warning: package 'tidyr' was built under R version 4.3.3

Warning: package 'readr' was built under R version 4.3.3

Warning: package 'purrr' was built under R version 4.3.3

Warning: package 'dplyr' was built under R version 4.3.2

Warning: package 'stringr' was built under R version 4.3.2

Warning: package 'lubridate' was built under R version 4.3.3

── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
✔ dplyr     1.1.4     ✔ readr     2.1.5
✔ forcats   1.0.0     ✔ stringr   1.5.1
✔ ggplot2   3.5.2     ✔ tibble    3.2.1
✔ lubridate 1.9.4     ✔ tidyr     1.3.1
✔ purrr     1.0.4     
── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()
ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors

library(readr)
library(scales)   # for label_number()

Warning: package 'scales' was built under R version 4.3.2

Attaching package: 'scales'

The following object is masked from 'package:purrr':

    discard

The following object is masked from 'package:readr':

    col_factor

# --- metadata (CTCF) ---
metadata <- tribble(
  ~Sample,           ~Sample_Det,
  "MCW_SP_ChIP63",   "Ind1_VEH_CTCF",
  "MCW_SP_ChIP64",   "Ind1_DOX_CTCF",
  "MCW_SP_ChIP65",   "Ind2_VEH_CTCF",
  "MCW_SP_ChIP66",   "Ind2_DOX_CTCF",
  "MCW_SP_ChIP67",   "Ind3_VEH_CTCF",
  "MCW_SP_ChIP68",   "Ind3_DOX_CTCF"
) |> mutate(
  Sample     = factor(Sample,     levels = Sample),
  Sample_Det = factor(Sample_Det, levels = Sample_Det)
)

# --- load all cutoff-analysis files (CTCF narrow peaks) ---
files <- list.files("data/macs3_narrow_out_CTCF",
                    pattern = "_cutoff_analysis\\.txt$", full.names = TRUE)

df <- files |>
  set_names() |>
  map_dfr(~ read_delim(.x, delim = "\t", show_col_types = FALSE), .id = "filepath") |>
  mutate(Sample = basename(filepath) |> str_remove("_cutoff_analysis\\.txt")) |>
  left_join(metadata, by = "Sample") |>
  mutate(Sample_Det = factor(Sample_Det, levels = levels(metadata$Sample_Det))) |>
  arrange(Sample_Det, qscore)

# Common x-scale (guarantees tick at 0 through 7)
# Common x-axis (ticks only; no limits here)
x_ticks <- scale_x_continuous(
  breaks = 0:7,
  labels = as.character(0:7),
  expand = c(0, 0)
)

# ---- Plot with linear y-axis ----
p_linear <- ggplot(df, aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks +
  coord_cartesian(xlim = c(0, 7)) +   # zoom without dropping rows
  scale_y_continuous(labels = scales::label_number(big.mark = ",")) +
  labs(x = "Q Score", y = "Peak Counts (linear)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank())

Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
ℹ Please use `linewidth` instead.
This warning is displayed once every 8 hours.
Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
generated.

# ---- Plot with log10 y-axis ----
p_log <- df %>%
  mutate(npeaks = ifelse(npeaks <= 0, NA_real_, npeaks)) %>%  # log can't show 0/neg
  ggplot(aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +    # drop NA quietly
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks +
  coord_cartesian(xlim = c(0, 7)) +                           # no row removal
  scale_y_log10(labels = scales::label_number(big.mark = ",")) +
  labs(x = "Q Score", y = "Peak Counts (log10)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank())

p_linear

p_log

📌 RAD21 peaks cutoff (before deduplication)

library(tidyverse)
library(readr)
library(scales)   # for label_number()

# --- metadata (RAD21) ---
metadata <- tribble(
  ~Sample,           ~Sample_Det,
  "MCW_SP_ChIP69",   "Ind1_VEH_RAD21",
  "MCW_SP_ChIP70",   "Ind1_DOX_RAD21",
  "MCW_SP_ChIP71",   "Ind2_VEH_RAD21",
  "MCW_SP_ChIP72",   "Ind2_DOX_RAD21",
  "MCW_SP_ChIP73",   "Ind3_VEH_RAD21",
  "MCW_SP_ChIP74",   "Ind3_DOX_RAD21"
) |>
  mutate(
    Sample     = factor(Sample,     levels = Sample),
    Sample_Det = factor(Sample_Det, levels = Sample_Det)
  )

# --- load cutoff-analysis files (RAD21 narrow peaks) ---
files <- list.files("data/macs3_narrow_out_RAD21",
                    pattern = "_cutoff_analysis\\.txt$", full.names = TRUE)

df <- files |>
  set_names() |>
  map_dfr(~ read_delim(.x, delim = "\t", show_col_types = FALSE), .id = "filepath") |>
  mutate(Sample = basename(filepath) |> str_remove("_cutoff_analysis\\.txt")) |>
  left_join(metadata, by = "Sample") |>
  mutate(Sample_Det = factor(Sample_Det, levels = levels(metadata$Sample_Det))) |>
  arrange(Sample_Det, qscore)

# X-axis ticks (0..7) and view window without dropping rows
x_ticks <- scale_x_continuous(breaks = 0:7, labels = as.character(0:7), expand = c(0, 0))

# ---- Plot with linear y-axis ----
p_linear <- ggplot(df, aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks + coord_cartesian(xlim = c(0, 7)) +
  scale_y_continuous(labels = scales::label_number(big.mark = ",")) +
  labs(title = "RAD21 narrow peaks across samples (before deduplication)",
       x = "Q Score", y = "Peak Counts (linear)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank(),
        plot.title = element_text(hjust = 0.5, face = "bold"))

# ---- Plot with log10 y-axis ----
p_log <- df |>
  mutate(npeaks = ifelse(npeaks <= 0, NA_real_, npeaks)) |>
  ggplot(aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks + coord_cartesian(xlim = c(0, 7)) +
  scale_y_log10(labels = scales::label_number(big.mark = ",")) +
  labs(title = "RAD21 narrow peaks across samples (before deduplication)",
       x = "Q Score", y = "Peak Counts (log10)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank(),
        plot.title = element_text(hjust = 0.5, face = "bold"))

# ---- Print both ----
p_linear

p_log

📌 CTCF peaks cutoff (after deduplication)

library(tidyverse)
library(readr)
library(scales)   # for label_number()

# --- metadata (CTCF) ---
metadata <- tribble(
  ~Sample,           ~Sample_Det,
  "MCW_SP_ChIP63",   "Ind1_VEH_CTCF",
  "MCW_SP_ChIP64",   "Ind1_DOX_CTCF",
  "MCW_SP_ChIP65",   "Ind2_VEH_CTCF",
  "MCW_SP_ChIP66",   "Ind2_DOX_CTCF",
  "MCW_SP_ChIP67",   "Ind3_VEH_CTCF",
  "MCW_SP_ChIP68",   "Ind3_DOX_CTCF"
) |> mutate(
  Sample     = factor(Sample,     levels = Sample),
  Sample_Det = factor(Sample_Det, levels = Sample_Det)
)

# --- load all cutoff-analysis files (CTCF narrow peaks) ---
files <- list.files("data/macs3_narrow_out_CTCF_dedup",
                    pattern = "_cutoff_analysis\\.txt$", full.names = TRUE)

df <- files |>
  set_names() |>
  map_dfr(~ read_delim(.x, delim = "\t", show_col_types = FALSE), .id = "filepath") |>
  mutate(Sample = basename(filepath) |> str_remove("_cutoff_analysis\\.txt")) |>
  left_join(metadata, by = "Sample") |>
  mutate(Sample_Det = factor(Sample_Det, levels = levels(metadata$Sample_Det))) |>
  arrange(Sample_Det, qscore)

# Common x-scale (guarantees tick at 0 through 7)
# Common x-axis (ticks only; no limits here)
x_ticks <- scale_x_continuous(
  breaks = 0:7,
  labels = as.character(0:7),
  expand = c(0, 0)
)

# ---- Plot with linear y-axis ----
p_linear <- ggplot(df, aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks +
  coord_cartesian(xlim = c(0, 7)) +   # zoom without dropping rows
  scale_y_continuous(labels = scales::label_number(big.mark = ",")) +
  labs(x = "Q Score", y = "Peak Counts (linear)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank())

# ---- Plot with log10 y-axis ----
p_log <- df %>%
  mutate(npeaks = ifelse(npeaks <= 0, NA_real_, npeaks)) %>%  # log can't show 0/neg
  ggplot(aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +    # drop NA quietly
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks +
  coord_cartesian(xlim = c(0, 7)) +                           # no row removal
  scale_y_log10(labels = scales::label_number(big.mark = ",")) +
  labs(x = "Q Score", y = "Peak Counts (log10)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank())

p_linear

p_log

📌 RAD21 peaks cutoff (after deduplication)

library(tidyverse)
library(readr)
library(scales)   # for label_number()

# --- metadata (RAD21) ---
metadata <- tribble(
  ~Sample,           ~Sample_Det,
  "MCW_SP_ChIP69",   "Ind1_VEH_RAD21",
  "MCW_SP_ChIP70",   "Ind1_DOX_RAD21",
  "MCW_SP_ChIP71",   "Ind2_VEH_RAD21",
  "MCW_SP_ChIP72",   "Ind2_DOX_RAD21",
  "MCW_SP_ChIP73",   "Ind3_VEH_RAD21",
  "MCW_SP_ChIP74",   "Ind3_DOX_RAD21"
) |>
  mutate(
    Sample     = factor(Sample,     levels = Sample),
    Sample_Det = factor(Sample_Det, levels = Sample_Det)
  )

# --- load cutoff-analysis files (RAD21 narrow peaks) ---
files <- list.files("data/macs3_narrow_out_RAD21_dedup",
                    pattern = "_cutoff_analysis\\.txt$", full.names = TRUE)

df <- files |>
  set_names() |>
  map_dfr(~ read_delim(.x, delim = "\t", show_col_types = FALSE), .id = "filepath") |>
  mutate(Sample = basename(filepath) |> str_remove("_cutoff_analysis\\.txt")) |>
  left_join(metadata, by = "Sample") |>
  mutate(Sample_Det = factor(Sample_Det, levels = levels(metadata$Sample_Det))) |>
  arrange(Sample_Det, qscore)

# X-axis ticks (0..7) and view window without dropping rows
x_ticks <- scale_x_continuous(breaks = 0:7, labels = as.character(0:7), expand = c(0, 0))

# ---- Plot with linear y-axis ----
p_linear <- ggplot(df, aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks + coord_cartesian(xlim = c(0, 7)) +
  scale_y_continuous(labels = scales::label_number(big.mark = ",")) +
  labs(title = "RAD21 narrow peaks across samples (after deduplication)",
       x = "Q Score", y = "Peak Counts (linear)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank(),
        plot.title = element_text(hjust = 0.5, face = "bold"))

# ---- Plot with log10 y-axis ----
p_log <- df |>
  mutate(npeaks = ifelse(npeaks <= 0, NA_real_, npeaks)) |>
  ggplot(aes(qscore, npeaks, group = Sample)) +
  geom_line(size = 0.8, color = "#d95f02", na.rm = TRUE) +
  facet_wrap(~ Sample_Det, scales = "free_y", ncol = 3) +
  x_ticks + coord_cartesian(xlim = c(0, 7)) +
  scale_y_log10(labels = scales::label_number(big.mark = ",")) +
  labs(title = "RAD21 narrow peaks across samples (after deduplication)",
       x = "Q Score", y = "Peak Counts (log10)") +
  theme_minimal(base_size = 12) +
  theme(strip.text = element_text(face = "bold", size = 10),
        panel.grid.minor = element_blank(),
        plot.title = element_text(hjust = 0.5, face = "bold"))

# ---- Print both ----
p_linear

p_log

Session information

sessionInfo()

R version 4.3.0 (2023-04-21 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 11 x64 (build 26100)

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] scales_1.3.0    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

loaded via a namespace (and not attached):
 [1] sass_0.4.10       generics_0.1.3    stringi_1.8.3     hms_1.1.3        
 [5] digest_0.6.34     magrittr_2.0.3    evaluate_1.0.3    grid_4.3.0       
 [9] timechange_0.3.0  fastmap_1.2.0     rprojroot_2.0.4   workflowr_1.7.1  
[13] jsonlite_2.0.0    promises_1.3.2    jquerylib_0.1.4   cli_3.6.1        
[17] crayon_1.5.3      rlang_1.1.3       bit64_4.6.0-1     munsell_0.5.1    
[21] withr_3.0.2       cachem_1.1.0      yaml_2.3.10       parallel_4.3.0   
[25] tools_4.3.0       tzdb_0.5.0        colorspace_2.1-0  httpuv_1.6.15    
[29] vctrs_0.6.5       R6_2.6.1          lifecycle_1.0.4   git2r_0.36.2     
[33] bit_4.6.0         fs_1.6.3          vroom_1.6.5       pkgconfig_2.0.3  
[37] pillar_1.10.2     bslib_0.9.0       later_1.3.2       gtable_0.3.6     
[41] glue_1.7.0        Rcpp_1.0.12       xfun_0.52         tidyselect_1.2.1 
[45] rstudioapi_0.17.1 knitr_1.50        farver_2.1.2      htmltools_0.5.8.1
[49] labeling_0.4.3    rmarkdown_2.29    compiler_4.3.0