RSCU (relative synonymous codon usage) values of multiple DNA sequences can be analyzed using the RSCUcaller package. The package offers functions to retrieve RSCU information, plot the values, and calculate statistical significance.
The "inst/extdata" folder contains sample data in the form of tables and FASTA files.
install.packages("devtools")
library(devtools)
devtools::install_github('Mordziarz/RSCUcaller')
library(RSCUcaller)Install RSCUcaller from Bioconductor (awaiting review by Bioconductor):
if (!requireNamespace("BiocManager", quietly=TRUE))
install.packages("BiocManager")
BiocManager::install("RSCUcaller")Install missing CRAN packages
install.packages(setdiff(c("stats", "dplyr", "ggplot2",
"ggpubr", "seqinr","rstatix",
"patchwork", "forcats", "phylogram",
"circlize", "smplot2", "stringr"),
installed.packages()[,"Package"]))Load CRAN packages
lapply(c("stats", "dplyr", "ggplot2",
"ggpubr", "seqinr","rstatix",
"patchwork", "forcats", "phylogram",
"circlize", "smplot2", "stringr"), library, character.only = TRUE)Install BiocManager if not installed
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")Install missing Bioconductor packages
BiocManager::install(setdiff(c("ComplexHeatmap","ggtree"),
installed.packages()[,"Package"]))Load Bioconductor packages
lapply(c("ComplexHeatmap","ggtree"),
library, character.only = TRUE)To use all features of the program, you will need several libraries.
library(ggplot2)
library(dplyr)
library(seqinr)
library(rstatix)
library(ggpubr)
library(patchwork)
library(forcats)
library(phylogram)
library(circlize)
library(ComplexHeatmap)
library(smplot2)
library(ggtree)
library(stats)
library(stringr)set.seed(123)Tables and graphics are saved to folders by the RSCUcaller package. To specify the desired output directory, it is recommended to use the setwd() function.
setwd("path/to/your/directory")RSCUcaller requires DNA sequences from NCBI. To begin the analysis, the FASTA file must be prepared appropriately using the prepare_fasta() function. This function requires a table with two columns:
"sequence_path" - This column specifies the path to the FASTA file.
"sample_name" - This column defines the FASTA name, which will be displayed on visualizations and should start with a number followed by an underscore (e.g., 1_Riccia_fluitans).
The "samples_table" should resemble the following. You can create this using a program other than R, such as a simple text editor and then use the read.csv2() function in R.
Ensure that the column names match the column names in the tutorial.
Please ensure that the sample names ("sample_name") and "GENBANK_ACCESSION" are unique. The program requires the names to be as detailed as possible. For example, if the user provides the names "1_A2" and "2_A2.1", the program will treat them as a single sample. However, in the case of names "1_A2.1" and "2_A2.2", the program will function correctly.
When you have FASTA files in separate files
| sequence_path | sample_name |
|---|---|
| path/to/your/fasta | 1_fasta_name |
| path/to/your/fasta | 2_fasta_name |
| path/to/your/fasta | 3_fasta_name |
| path/to/your/fasta | 4_fasta_name |
| path/to/your/fasta | 5_fasta_name |
| path/to/your/fasta | 6_fasta_name |
| path/to/your/fasta | 7_fasta_name |
| path/to/your/fasta | 8_fasta_name |
path1 <- "/path/to/your/fasta"
samples_table <- data.frame(sequence_path = c(path1,path2),
sample_name = c("1_fasta_name","2_fasta_name"))
prepare_fasta(samples_table = samples_table,file_out = "your_fasta.fasta")Alternatively, if multiple species from NCBI were downloaded within a single FASTA file, the samples_table with the ID and GENBANK_ACCESSION columns can be used. In this scenario, the path to the FASTA file must also be provided using the 8000 path argument.
When you have downloaded multiple FASTA files from NCBI into a single file
| ID | GENBANK_ACCESSION |
|---|---|
| 1_fasta_name | gene_bank_accession_id_1 |
| 2_fasta_name | gene_bank_accession_id_2 |
| 3_fasta_name | gene_bank_accession_id_3 |
| 4_fasta_name | gene_bank_accession_id_4 |
| 5_fasta_name | gene_bank_accession_id_5 |
| 6_fasta_name | gene_bank_accession_id_6 |
| 7_fasta_name | gene_bank_accession_id_7 |
| 8_fasta_name | gene_bank_accession_id_8 |
samples_table <- data.frame(ID = c("1_fasta_name","2_fasta_name"),
GENBANK_ACCESSION = c("gene_bank_accession_id_1","gene_bank_accession_id_2"))
prepare_fasta(samples_table = samples_table, path = "/path/to/multiple/sequence/fasta", file_out = "your_fasta.fasta")The name of the prepared FASTA file needs to be specified regardless of the chosen method. This file will be created and saved in your working directory using the argument file_out="name_of_your_output.fasta".
The main function of the package, get_RSCU(), can be used to calculate RSCU values directly from a set of previously prepared sequences.
get_RSCU_out <- get_RSCU(merged_sequences = "your_fasta.fasta")The program implements a function that performs RSCU analysis for alternative genetic codes by modifying the encoding of certain amino acids. A table is available containing codon identifiers (codon_table_id) that are required to use the get_RSCU_other() function.
| codon_table_id | Genetic Code Description |
|---|---|
| 1 | Standard |
| 2 | Vertebrate Mitochondrial |
| 3 | Yeast Mitochondrial |
| 4 | Mold Mitochondrial; Protozoan Mitochondrial; Coelenterate Mitochondrial; Mycoplasma; Spiroplasma |
| 5 | Invertebrate Mitochondrial |
| 6 | Ciliate Nuclear; Dasycladacean Nuclear; Hexamita Nuclear |
| 9 | Echinoderm Mitochondrial; Flatworm Mitochondrial |
| 10 | Euplotid Nuclear |
| 11 | Bacterial, Archaeal and Plant Plastid |
| 12 | Alternative Yeast Nuclear |
| 13 | Ascidian Mitochondrial |
| 14 | Alternative Flatworm Mitochondrial |
| 15 | Blepharisma Macronuclear |
| 16 | Chlorophycean Mitochondrial |
| 21 | Trematode Mitochondrial |
| 22 | Scenedesmus obliquus Mitochondrial |
| 23 | Thraustochytrium Mitochondrial |
| 24 | Pterobranchia Mitochondrial |
| 25 | Candidate Division SR1 and Gracilibacteria |
| 26 | Pachysolen tannophilus Nuclear |
The user can check which codons encode a specific amino acid in a given codon_table_id by using the get_codon_table() function.
get_codon_table(codon_table_id = 1)Two functions have been created to facilitate codon analysis with other genetic codes.
If you are only analyzing Vertebrate mitochondrial genomes, you can simply use the get_RSCU_other() function.
get_RSCU_other(merged_sequences = "prepered_fasta.fasta",codon_table_id = 2,pseudo_count = 0)However, if you want to compare different genetic codes, just add a column named codon_table_id to your samples_table and use the get_RSCU_other2() function.
| sequence_path | sample_name | codon_table_id |
|---|---|---|
| path/to/your/fasta | 1_fasta_name | 2 |
| path/to/your/fasta | 2_fasta_name | 2 |
| path/to/your/fasta | 3_fasta_name | 3 |
| path/to/your/fasta | 4_fasta_name | 4 |
| path/to/your/fasta | 5_fasta_name | 5 |
| path/to/your/fasta | 6_fasta_name | 6 |
| path/to/your/fasta | 7_fasta_name | 2 |
| path/to/your/fasta | 8_fasta_name | 2 |
or
| ID | GENBANK_ACCESSION | codon_table_id |
|---|---|---|
| 1_fasta_name | gene_bank_accession_id_1 | 2 |
| 2_fasta_name | gene_bank_accession_id_2 | 3 |
| 3_fasta_name | gene_bank_accession_id_3 | 2 |
| 4_fasta_name | gene_bank_accession_id_4 | 4 |
| 5_fasta_name | gene_bank_accession_id_5 | 2 |
| 6_fasta_name | gene_bank_accession_id_6 | 2 |
| 7_fasta_name | gene_bank_accession_id_7 | 2 |
| 8_fasta_name | gene_bank_accession_id_8 | 2 |
get_RSCU_other2(merged_sequences = "prepered_fasta.fasta",samples_table=samples_table,pseudo_count = 0)The get_matrix() function allows the user to create a matrix that the user can use in any way they like. Simply use the result of the get_RSCU() function. The user will be able to use the matrices from this function, for example, in generating PCA.
get_matrix(get_RSCU_out = get_RSCU_out)A heatmap and a dendrogram can be drawn using the heatmap_RSCU() function. The heatmap is generated from the output of the get_RSCU() function. To create the heatmap, specify "heatmap" in the select argument and choose a color scheme from "red_green", "green_red", "blue_green", "green_blue", "blue_red", or "red_blue".
heatmap_RSCU(get_RSCU_out = get_RSCU_out, select = "heatmap", heatmap_color = "red_blue")A dendrogram can be obtained by providing the output of the get_RSCU() function and specifying "dendrogram" in the select argument. The result can be further edited using packages like ggtree. The dendrogram will also be saved as a file in your working directory named dendrogram_from_heatmap.newick by the system.
heatmap_RSCU(get_RSCU_out = get_RSCU_out, select = "dendogram")Histograms can be created from the output of the get_RSCU() function using our package.
histogram_RSCU(get_RSCU_out = get_RSCU_out, title = "graph title")Double histograms are plotted using the histogram_RSCU_double() function. This function requires two input parameters: get_RSCU_out_left and get_RSCU_out_right. Additionally, two optional title parameters, title_left and title_right, can be provided.
histogram_RSCU_double(get_RSCU_out_left = get_RSCU_out, get_RSCU_out_right = get_RSCU_out, title_left = "left title", title_right = "right title")Pearson correlation between two species can be performed using the RSCUcaller package. The names of the Species columns in the get_RSCU_out data frame to be correlated should be specified.
correlation(get_RSCU_out = get_RSCU_out, Species_x = "Species_x", Species_y = "Species_y", xlab = "title of x lab", ylab = "title of y lab")Statistical analysis of RSCU values at the codon level can be performed using the boxplot_between_groups() function. This function utilizes the Kruskal-Wallis test, Welch ANOVA, and ANOVA to assess significant differences in RSCU values between groups, and subsequently the Dunn's post-hoc test, Pairwise t-test, and TukeyHSD for pairwise comparisons.
To utilize this function, a table named grouping_table needs to be prepared with the following two columns:
"Species"-This column should contain the names of the sequences, corresponding to the Species column from the get_RSCU_out() data frame.
"group"-This column should specify the group to which each sequence belongs.
The function will generate graphical outputs. These outputs will be placed in two folders: selected_species and selected_species_barplots. Additionally, a table named Post_hoc_table_selected_species.csv will be created in the working directory.
The "grouping_table" should look like this:
Ensure that the column names match the column names in the tutorial.
| Species | group |
|---|---|
| Species1 | group1 |
| Species2 | group2 |
| Species3 | group3 |
| Species4 | group4 |
| Species5 | group1 |
| Species6 | group1 |
| Species7 | group2 |
| Species8 | group3 |
boxplot_between_groups(get_RSCU_out = get_RSCU_out, grouping_table = grouping_table, width = 6, height = 6, xlab = "title of x lab", res = 300,p.adjust.method = "bonferroni")neutrality_pr2(get_RSCU_out = get_RSCU_out,select = "PR2_plot",grouping_table = grouping_table)neutrality_pr2(get_RSCU_out = get_RSCU_out,select = "neutrality_plot",grouping_table = grouping_table)PCA_RSCU(get_matrix_out = get_matrix_out,grouping_table = grouping_table)Statistical analysis and visualizations can be generated for your data using the stat_scat_box() function. This function utilizes the Kruskal-Wallis test, Welch ANOVA, and ANOVA to assess significant differences in RSCU values between groups, and subsequently the Dunn's post-hoc test, Pairwise t-test, and TukeyHSD for pairwise comparisons. A table named Post_hoc_table_aminoacids.csv and three folders, boxplots, barplots and scatter_plots, will also be created in your working directory.
stat_scat_box(get_RSCU_out = get_RSCU_out, width = 6, height = 6, res = 300,p.adjust.method = "bonferroni")When utilizing RSCUcaller, kindly cite: Maździarz, M., Zając, S., Paukszto, Ł. et al. RSCUcaller: an R package for analyzing differences in relative synonymous codon usage (RSCU). BMC Bioinformatics 26, 141 (2025). https://doi.org/10.1186/s12859-025-06166-5
Any issues connected with the RSCUcaller should be addressed to Mateusz Mazdziarz (mateusz.mazdziarz@uwm.edu.pl).
https://doi.org/10.3390/genes15050562
https://doi.org/10.1038/s41598-023-35269-3
- Please check your FASTA sequences to ensure each one runs from the start codon to the stop codon, remembering to 'reverse' the CDS
- Please ensure that the sample names ("sample_name") and "GENBANK_ACCESSION" are unique. The program requires the names to be as detailed as possible. For example, if the user provides the names "1_A2" and "2_A2.1", the program will treat them as a single sample. However, in the case of names "1_A2.1" and "2_A2.2", the program will function correctly.
- Nucleotides other than ATCG, such as U or IUPAC, will be skipped by the program and will skew the results.
- Program reading of nucleotides other than ATCG
- Ability to perform RSCU analysis on RNA by adding expression as an additional dimension