Total Reads and Mapped Reads

Last updated: 2025-01-30

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File	Version	Author	Date	Message
Rmd	e3b04c6	sayanpaul01	2025-01-30	Added Total Reads and Mapped Reads visualization

📌 Total Reads by Sample

This section visualizes the total RNA-sequencing reads across samples.

Load Required Libraries

# Load necessary R packages
library(limma)

Warning: package 'limma' was built under R version 4.3.1

library(RColorBrewer)
library(data.table)

Warning: package 'data.table' was built under R version 4.3.2

library(tidyverse)

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

Warning: package 'ggplot2' was built under R version 4.3.3

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 4.3.1

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 4.3.1

── 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   3.5.1     ✔ tibble    3.2.1
✔ lubridate 1.9.3     ✔ tidyr     1.3.1
✔ purrr     1.0.2     
── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::between()     masks data.table::between()
✖ dplyr::filter()      masks stats::filter()
✖ dplyr::first()       masks data.table::first()
✖ lubridate::hour()    masks data.table::hour()
✖ lubridate::isoweek() masks data.table::isoweek()
✖ dplyr::lag()         masks stats::lag()
✖ dplyr::last()        masks data.table::last()
✖ lubridate::mday()    masks data.table::mday()
✖ lubridate::minute()  masks data.table::minute()
✖ lubridate::month()   masks data.table::month()
✖ lubridate::quarter() masks data.table::quarter()
✖ lubridate::second()  masks data.table::second()
✖ purrr::transpose()   masks data.table::transpose()
✖ lubridate::wday()    masks data.table::wday()
✖ lubridate::week()    masks data.table::week()
✖ lubridate::yday()    masks data.table::yday()
✖ lubridate::year()    masks data.table::year()
ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors

library(scales)

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

library(ggplot2)
library(dplyr)

📍 2. Load Data

# Load the dataset containing the total reads per sample
align <- read.csv("data/Total_number_of_reads_by_sample.csv")  # Ensure the file is in the 'data/' folder
map <- data.frame(align)

📍 3. Define Color Palettes

# Define color palettes for plots
drug_palc <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
Ind_palc <- c("#ffbe0b","#ff006e","#fb5607", "#8338ec","#3a86ff","#4a4e69")
Time_palc <- c("#0000FF","#80FF00", "#FF00FF")
Combined_palc <- c("#FF0000","#00FF00","#0000FF","#FFFF00","#FF00FF","#00FFFF",
                   "#FFA500","#800080","#FFC0CB","#A52A2A","#808080","#FFD700",
                   "#008080","#000080","#FFFFFF","#000000","#D2691E","#ADFF2F")

📍 4. Prepare Data

# Factor Sample_name to maintain order
map$Sample_name <- factor(map$Sample_name, levels = map$Sample_name)

📍 5. Plot Total Reads by Sample

# Generate the bar plot
p <- ggplot(map, aes(x = Sample_name, y = Counts, fill = Condition)) +
  geom_col() +
  scale_fill_manual(values = drug_palc) +
  scale_y_continuous(labels = function(x) paste0(x / 1e6, "M")) +
  ggtitle(expression("Total number of reads by sample")) +
  xlab("") +
  ylab(expression("RNA-sequencing reads")) +
  theme_bw() +
  theme(
    plot.title = element_text(size = rel(2), hjust = 0.5),
    axis.title = element_text(size = 15, color = "black"),
    axis.ticks = element_line(linewidth = 1.5),
    axis.line = element_line(linewidth = 1.5),
    axis.text.y = element_text(size = 10, color = "black", angle = 0, hjust = 0.8, vjust = 0.5),
    axis.text.x = element_text(size = 10, color = "black", angle = 90, hjust = 1, vjust = 0.2)
  )

# Save the plot as an image
ggsave("output/total_reads_by_sample_plot.png", p)

# Display the plot in the document
p

📌 Total Reads by Treatment

📍 Define Color Palettes

# Define color palettes for plots
drug_palc <- c("#8B006D","#DF707E","#F1B72B", "#3386DD","#707031","#41B333")
Ind_palc <- c("#ffbe0b","#ff006e","#fb5607", "#8338ec","#3a86ff","#4a4e69")
Time_palc <- c("#0000FF","#80FF00", "#FF00FF")
Combined_palc <- c("#FF0000","#00FF00","#0000FF","#FFFF00","#FF00FF","#00FFFF",
                   "#FFA500","#800080","#FFC0CB","#A52A2A","#808080","#FFD700",
                   "#008080","#000080","#FFFFFF","#000000","#D2691E","#ADFF2F")

📍 Load dataset for total reads by treatment

align1 <- read.csv("data/Total_number_of_reads_by_treatment.csv")  # Ensure this file is inside the 'data/' folder
map1 <- data.frame(align1)

📍 Generate the boxplot

p_treatment <- ggplot(map1, aes(x = Condition, y = Counts, fill = Condition)) +
  geom_boxplot() +
  scale_fill_manual(values = drug_palc) +
  scale_y_continuous(
    limits = c(0, 40000000),  # Set y-axis range
    labels = function(x) paste0(x / 1e6, "M")  # Display labels in millions
  ) +
  ggtitle(expression("Total number of reads by treatment")) +
  xlab("") +
  ylab(expression("RNA-sequencing reads")) +
  theme_bw() +
  theme(
    plot.title = element_text(size = rel(2), hjust = 0.5),
    axis.title = element_text(size = 15, color = "black"),
    axis.text.x = element_text(size = 10, angle = 90, hjust = 1, vjust = 0.2)
  )

# Save the plot as an image
ggsave("output/total_reads_by_treatment_plot.png", p_treatment)

# Display the plot in the document
p_treatment

📌 Total Reads by Individuals

📍 Define Color Palettes

# Define color palette for individuals
Ind_palc <- c("#ffbe0b","#ff006e","#fb5607", "#8338ec","#3a86ff","#4a4e69")

📍 Load dataset for total reads by individual

# Load dataset for total reads by individual
align2 <- read.csv("data/Total_number_of_reads_by_Individuals.csv")  # Ensure this file is inside the 'data/' folder
map2 <- data.frame(align2)

📍 Generate the boxplot

# Generate the boxplot
p_individual <- ggplot(map2, aes(x = Ind, y = Counts, fill = Individual)) +
  geom_boxplot() +
  scale_fill_manual(values = Ind_palc) +
  scale_y_continuous(
    limits = c(0, 40000000),  # Set y-axis range
    labels = function(x) paste0(x / 1e6, "M")  # Display labels in millions
  ) +
  ggtitle(expression("Total number of reads by individual")) +
  xlab("") +
  ylab(expression("RNA-sequencing reads")) +
  theme_bw() +
  theme(
    plot.title = element_text(size = rel(2), hjust = 0.5),
    axis.title = element_text(size = 15, color = "black"),
    axis.text.x = element_text(size = 10, angle = 90, hjust = 1, vjust = 0.2)
  )

# Save the plot as an image
ggsave("output/total_reads_by_individual_plot.png", p_individual)

# Display the plot in the document
p_individual

📌 Total Reads by Time

📍 Define Color Palettes

# Define color palette for time points
Time_palc <- c("#0000FF","#80FF00", "#FF00FF")

📍 Load dataset for total reads by time

# Load dataset for total reads by time
align3 <- read.csv("data/Total_number_of_reads_by_time.csv")  # Ensure this file is inside the 'data/' folder
map3 <- data.frame(align3)

📍 Generate the boxplot

# Generate the boxplot
p_time <- ggplot(map3, aes(x = Condition, y = Counts, fill = Time)) +
  geom_boxplot() +
  scale_fill_manual(values = Time_palc) +
  scale_y_continuous(
    limits = c(0, 40000000),  # Set y-axis range
    labels = function(x) paste0(x / 1e6, "M")  # Display labels in millions
  ) +
  ggtitle(expression("Total number of reads by time")) +
  xlab("") +
  ylab(expression("RNA-sequencing reads")) +
  theme_bw() +
  theme(
    plot.title = element_text(size = rel(2), hjust = 0.5),
    axis.title = element_text(size = 15, color = "black"),
    axis.text.x = element_text(size = 10, angle = 90, hjust = 1, vjust = 0.2)
  )

# Save the plot as an image
ggsave("output/total_reads_by_time_plot.png", p_time)

# Display the plot in the document
p_time

sessionInfo()

R version 4.3.0 (2023-04-21 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 11 x64 (build 22631)

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] ggpubr_0.6.0       Hmisc_5.2-0        corrplot_0.95      ggrepel_0.9.6     
 [5] cowplot_1.1.3      biomaRt_2.58.2     reshape2_1.4.4     gridExtra_2.3     
 [9] edgeR_4.0.1        scales_1.3.0       lubridate_1.9.3    forcats_1.0.0     
[13] stringr_1.5.1      dplyr_1.1.4        purrr_1.0.2        readr_2.1.5       
[17] tidyr_1.3.1        tibble_3.2.1       ggplot2_3.5.1      tidyverse_2.0.0   
[21] data.table_1.14.10 RColorBrewer_1.1-3 limma_3.58.1       workflowr_1.7.1   

loaded via a namespace (and not attached):
  [1] DBI_1.2.3               bitops_1.0-7            rlang_1.1.3            
  [4] magrittr_2.0.3          git2r_0.35.0            compiler_4.3.0         
  [7] RSQLite_2.3.3           getPass_0.2-4           png_0.1-8              
 [10] systemfonts_1.1.0       callr_3.7.6             vctrs_0.6.5            
 [13] pkgconfig_2.0.3         crayon_1.5.3            fastmap_1.1.1          
 [16] backports_1.5.0         dbplyr_2.5.0            XVector_0.42.0         
 [19] labeling_0.4.3          promises_1.3.0          rmarkdown_2.29         
 [22] tzdb_0.4.0              ps_1.8.1                ragg_1.3.3             
 [25] bit_4.0.5               xfun_0.50               zlibbioc_1.48.0        
 [28] cachem_1.0.8            GenomeInfoDb_1.38.8     jsonlite_1.8.9         
 [31] progress_1.2.3          blob_1.2.4              later_1.3.2            
 [34] broom_1.0.7             cluster_2.1.6           prettyunits_1.2.0      
 [37] R6_2.5.1                bslib_0.8.0             stringi_1.8.3          
 [40] car_3.1-3               rpart_4.1.23            jquerylib_0.1.4        
 [43] Rcpp_1.0.12             knitr_1.49              base64enc_0.1-3        
 [46] IRanges_2.36.0          nnet_7.3-19             httpuv_1.6.15          
 [49] timechange_0.3.0        tidyselect_1.2.1        abind_1.4-8            
 [52] rstudioapi_0.17.1       yaml_2.3.10             curl_6.0.1             
 [55] processx_3.8.5          lattice_0.22-5          plyr_1.8.9             
 [58] Biobase_2.62.0          withr_3.0.2             KEGGREST_1.42.0        
 [61] evaluate_1.0.3          foreign_0.8-87          BiocFileCache_2.10.2   
 [64] xml2_1.3.6              Biostrings_2.70.1       pillar_1.10.1          
 [67] filelock_1.0.3          carData_3.0-5           whisker_0.4.1          
 [70] checkmate_2.3.2         stats4_4.3.0            generics_0.1.3         
 [73] rprojroot_2.0.4         RCurl_1.98-1.13         S4Vectors_0.40.1       
 [76] hms_1.1.3               munsell_0.5.1           glue_1.7.0             
 [79] tools_4.3.0             ggsignif_0.6.4          locfit_1.5-9.8         
 [82] fs_1.6.3                XML_3.99-0.17           grid_4.3.0             
 [85] AnnotationDbi_1.64.1    colorspace_2.1-0        GenomeInfoDbData_1.2.11
 [88] htmlTable_2.4.3         Formula_1.2-5           cli_3.6.1              
 [91] rappdirs_0.3.3          textshaping_0.4.1       gtable_0.3.6           
 [94] rstatix_0.7.2           sass_0.4.9              digest_0.6.34          
 [97] BiocGenerics_0.48.1     htmlwidgets_1.6.4       farver_2.1.2           
[100] memoise_2.0.1           htmltools_0.5.8.1       lifecycle_1.0.4        
[103] httr_1.4.7              statmod_1.5.0           bit64_4.0.5

Total Reads and Mapped Reads

Your Name

2025-01-30

📌 Total Reads by Sample

Load Required Libraries

📍 2. Load Data

📍 3. Define Color Palettes

📍 4. Prepare Data

📍 5. Plot Total Reads by Sample

📌 Total Reads by Treatment

📍 Define Color Palettes

📍 Load dataset for total reads by treatment

📍 Generate the boxplot

📌 Total Reads by Individuals

📍 Define Color Palettes

📍 Load dataset for total reads by individual

📍 Generate the boxplot

📌 Total Reads by Time

📍 Define Color Palettes

📍 Load dataset for total reads by time

📍 Generate the boxplot