README.Rmd

---
output: github_document
---

```{r, include = FALSE}
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>",
  fig.path = "man/figures/README-",
  out.width = "100%"
)
```

# SplineOmics

![Version](https://img.shields.io/badge/version-0.1.1-blue) [![License: MIT](https://img.shields.io/badge/License-MIT-yellow.svg)](./LICENSE) ![Maintained? Yes](https://img.shields.io/badge/Maintained%3F-yes-brightgreen.svg) ![R CMD Check](https://img.shields.io/badge/R%20CMD%20check-passed-brightgreen) [![Docker](https://img.shields.io/badge/docker-pull-blue)](https://hub.docker.com/r/thomasrauter/splineomics) ![Dependencies](https://img.shields.io/badge/dependencies-19-blue) ![Platforms](https://img.shields.io/badge/platforms-all-brightgreen)

<img src="man/figures/hex_logo.png" align="left" width="65"/>

The R package `SplineOmics` finds the significant features (hits) of time-series -omics data by using splines and `limma` for hypothesis testing. It then clusters the hits based on the spline shape while showing all results in summary HTML reports.

The graphical abstract below shows the full workflow streamlined by `SplineOmics`:

![Graphical Abstract of SplineOmics Workflow](man/figures/SplineOmics_graphical_abstract.png)

## Table of Contents

-   [📘 Introduction](#-introduction)
-   [🔧 Installation](#-installation)
    -   [🐳 Docker Container](#-docker-container)
-   [▶️ Usage](#-usage)
    -   [🎓 Tutorial](#-tutorial)
    -   [📋 Details](#-details)
    -   [🧬 RNA-seq and Glycan Data](#-rna-seq-and-glycan-data)
-   [📦 Dependencies](#-dependencies)
-   [📚 Further Reading](#-further-reading)
-   [❓ Getting Help](#-getting-help)
-   [🤝 Contributing](#-contributing)
-   [💬 Feedback](#-feedback)
-   [📜 License](#-license)
-   [🎓 Citation](#-citation)
-   [🌟 Contributors](#-contributors)
-   [🙏 Acknowledgements](#-acknowledgements)

## 📘 Introduction

Welcome to `SplineOmics`, an R package designed to streamline the analysis of -omics time-series data, followed by automated HTML report generation.

### Is the SplineOmics package of use for me?

If you have -omics data over time, the package will help you to run `limma` with splines, decide on which parameters to use, perform the clustering, run GSEA and show result plots in HTML reports. Any time-series data that is a valid input to the `limma` package is also a valid input to the `SplineOmics` package (such as transcriptomics, proteomics, phosphoproteomics, metabolomics, glycan fractional abundances, etc.).

### What do I need precisely?

1.  **Data**: A data matrix where each row is a feature (e.g., protein, metabolite, etc.) and each column is a sample taken at a specific time. The data must have no NA values, should have normally distributed features and no dependence between the samples.

2.  **Meta**: A table with metadata on the columns/samples of the data matrix (e.g., batch, time point, etc.)

3.  **Annotation** (optional): A table with identifiers on the rows/features of the data matrix (e.g., gene and protein name).

### Capabilities

With `SplineOmics`, you can:

-   **Automatically perform exploratory data analysis:**

    The `explore_data()` function generates an HTML report, containing various plots, such as density, PCA, and correlation heatmap plots ([example report](https://csbg.github.io/SplineOmics_html_reports/explore_data_PTX.html)).

-   **Explore various limma splines hyperparameters:**

    Test combinations of hyperparameters, such as different datasets, `limma` design formulas, degrees of freedom, p-value thresholds, etc., using the `screen_limma_hyperparams()` function ([example report](https://csbg.github.io/SplineOmics_html_reports/Data_1_Design_1_vs_Data_1_Design_2_PTX.html) (along with the [encoding](https://csbg.github.io/SplineOmics_html_reports/hyperparams_screen_meta_table.html))).

-   **Perform limma spline analysis:**

    Use the `run_limma_splines()` function to perform the `limma` analysis with splines once the optimal hyperparameters are identified ([example report](https://csbg.github.io/SplineOmics_html_reports/create_limma_report_PTX.html)).

-   **Cluster significant features:**

    Cluster the significant features (hits) identified in the spline analysis with the `cluster_hits()` function ([example report](https://csbg.github.io/SplineOmics_html_reports/report_clustered_hits_PTX.html)).

-   **Run GSEA with clustered hits:**

    Perform gene set enrichment analysis (GSEA) using the clustered hits with the `create_gsea_report()` function ([example report](https://csbg.github.io/SplineOmics_html_reports/create_gsea_report_PTX.html)).

## 🔧 Installation

Follow the steps below to install the `SplineOmics` package from the GitHub repository into your R environment.

#### Prerequisites

-   Ensure **R** is installed on your system. If not, download and install it from [CRAN](https://cran.r-project.org/).
-   **RStudio** is recommended for a more user-friendly experience with R. Download and install RStudio from [posit.co](https://posit.co/download/rstudio-desktop/).

#### Installation Steps

> **Note for Windows Users:**

Note that some installation paths potentially are not writable on **Windows**. Therefore, it can be necessary to set up a library path and use that path for the installations:

``` r
custom_lib_path <- "C:/Rlibs"  # Replace with your desired path

# Create the directory if it doesn't exist
if (!dir.exists(custom_lib_path)) {
    dir.create(
      custom_lib_path,
      showWarnings = FALSE,
      recursive = TRUE
      )
}

# Set the library path to include the new directory
.libPaths(c(custom_lib_path, .libPaths()))

# Check if the new library path is added successfully
if (custom_lib_path %in% .libPaths()) {
    message("Library path set to: ", custom_lib_path)
} else {
    stop("Failed to set library path.")
}
```

Alternatively, you can run `RStudio` as administrator once for the installation (which is however generally not recommended, because it is a security risk).

> **Additional note for Windows Users:**

During the installation on Windows, you might see a message indicating that Rtools is not installed, which is typically required for compiling R packages from source. However, for this installation, Rtools is not necessary, and you can safely ignore this message.

1.  **Open RStudio** or your R console.

2.  **Install `BiocManager`** for Bioconductor dependencies (if not already installed)

``` r
install.packages(
  "BiocManager"
  # lib = custom_lib_path 
)
```

3.  **Install Bioconductor dependencies** separately using `BiocManager`

``` r
BiocManager::install(
  c("ComplexHeatmap", "limma", "variancePartition")
  # force = TRUE   # when encountering issues
  # lib = custom_lib_path 
)
```

4.  **Install** the **`remotes`** package for GitHub downloads (if not already installed)

``` r
install.packages(
  "remotes"
  # lib = custom_lib_path
)
```

5.  **Install** the **`SplineOmics`** package from GitHub and all its non-Bioconductor dependencies, using `remotes`

``` r
remotes::install_github(
  "csbg/SplineOmics",   # GitHub repository
  ref = "0.1.1",        # Specify the tag to install
  dependencies = TRUE,  # Install all dependencies
  upgrade = "always"    # Always upgrade dependencies
  # force = TRUE        # when encountering issues
  # lib = custom_lib_path 
)
```

6.  **Verify** the **installation** of the `SplineOmics` package

``` r
# Verify the installation of the SplineOmics package
if ("SplineOmics" %in% rownames(installed.packages())) {
  message("SplineOmics was installed successfully.")
} else {
  message("SplineOmics installation failed. Please check for errors during installation.")
}
```

#### Troubleshooting

If you encounter errors related to dependencies or package versions during installation, try updating your R and RStudio to the latest versions and repeat the installation steps.

For issues specifically related to the `SplineOmics` package, check the [Issues section](https://github.com/csbg/SplineOmics/issues) of the GitHub repository for similar problems or to post a new issue.

### 🐳 Docker Container

Alternatively, you can run your analysis in a `Docker` container. The underlying `Docker` image encapsulates the `SplineOmics` package together with the necessary environment and dependencies. This ensures higher levels of reproducibility because the analysis is carried out in a consistent environment, independent of the operating system and its custom configurations.

Please note that you must have the `Docker Engine` installed on your machine. For instructions on how to install it, consult the official [Docker Engine installation guide](https://docs.docker.com/engine/install/).

More information about `Docker` containers can be found on the [official Docker page](https://www.docker.com/resources/what-container/).

For instructions on downloading the image of the `SplineOmics` package and running the container, please refer to the [Docker instructions](https://csbg.github.io/SplineOmics/articles/Docker-instructions.html).

#### Troubleshooting

If you face "permission denied" issues on Linux distributions, check this [vignette](https://csbg.github.io/SplineOmics/articles/Docker_permission_denied.html).

## ▶️ Usage

### 🎓 Tutorial {#tutorial}

[This tutorial](https://csbg.github.io/SplineOmics/articles/get-started.html) covers a real CHO cell time-series proteomics example from start to end.

To open an R Markdown file of the **tutorial** in `RStudio`, run:

``` r
library(SplineOmics)
open_tutorial()  
```

To open an R Markdown file in `RStudio` containing a **template** for your own analysis, run:

``` r
library(SplineOmics)
open_template()
```

### 📋 Details {#details}

A detailed description of all arguments and outputs of all the functions in the package (exported and internal functions) can be found [here](https://csbg.github.io/SplineOmics/reference/).

#### Design `limma` design formula

A quick guide on how to design a `limma` design formula can be found [here](https://csbg.github.io/SplineOmics/articles/design_limma_design_formula.html).

An explanation of the three different `limma` results is [here](https://csbg.github.io/SplineOmics/articles/limma_result_categories.html).

### 🧬 RNA-seq and Glycan Data {#rna-seq-and-glycan-data}

#### RNA-seq data

Transcriptomics data must be preprocessed for `limma`. You need to provide an appropriate object, such as a `voom` object, in the `rna_seq_data` argument of the `SplineOmics` object (see [documentation](https://csbg.github.io/SplineOmics/reference/create_splineomics.html)). Along with this, the normalized matrix (e.g., the `$E` slot of the `voom` object) must be passed to the `data` argument. This allows flexibility in preprocessing; you can use any method you prefer as long as the final object and matrix are compatible with limma. One way to preprocess your RNA-seq data is by using the `preprocess_rna_seq_data()` function included in the `SplineOmics` package (see [documentation](https://csbg.github.io/SplineOmics/reference/preprocess_rna_seq_data.html)).

[Here](https://csbg.github.io/SplineOmics/articles/RNA-seq%20analysis.html) you can find an example analysis of RNA-seq data with the SplineOmics package.

#### Glycan fractional abundance data

The glycan fractional abundance data matrix, where each row represents a type of glycan and the columns correspond to timepoints, must be transformed before analysis. This preprocessing step is essential due to the compositional nature of the data. In compositional data, an increase in the abundance of one component (glycan) necessarily results in a decrease in others, introducing a dependency among the variables that can bias the analysis. One way to address this issue is by applying the Centered Log Ratio (CLR) transformation to the data with the clr function from the compositions package:

``` r
library(compositions)
clr_transformed_data <- clr(data_matrix)  # use as SplineOmics input
```

The results from clr transformed data can be harder to understand and interpret however. If you prefer ease of interpretation and are fine that the results contain some artifacts due to the compositional nature of the data, log2 transform your data instead and use that as input for the `SplineOmics` package.

``` r
log2_transformed_data <- log2(data_matrix)  # use as SplineOmics input
```

## 📦 Dependencies

The `SplineOmics` package relies on several other R packages for its functionality. Below is a list of dependencies that will automatically be installed along with `SplineOmics`. If you already have these packages installed, ensure they are up to date to avoid any compatibility issues.

-   **ComplexHeatmap** (\>= 2.18.0): For creating complex heatmaps with advanced features.
-   **base64enc** (\>= 0.1-3): For encoding/decoding base64.
-   **dendextend** (\>= 1.17.1): For extending `dendrogram` objects, allowing for easier manipulation of dendrograms.
-   **dplyr** (\>= 1.1.4): For data manipulation.
-   **ggplot2** (\>= 3.5.1): For creating elegant data visualizations using the grammar of graphics.
-   **ggrepel** (\>= 0.9.5): For better label placement in `ggplot2`.
-   **here** (\>= 1.0.1): For constructing paths to your project’s files.
-   **limma** (\>= 3.58.1): For linear models in microarray and RNA-seq analysis.
-   **openxlsx** (\>= 4.2.6.1): For reading, writing, and editing `.xlsx` files.
-   **patchwork** (\>= 1.2.0): For combining multiple `ggplot` objects into a single plot.
-   **pheatmap** (\>= 1.0.12): For creating aesthetically pleasing heatmaps.
-   **progress** (\>= 1.2.3): For adding progress bars to loops and apply functions.
-   **purrr** (\>= 1.0.2): For functional programming tools.
-   **rlang** (\>= 1.1.4): For working with core language features of R.
-   **scales** (\>= 1.3.0): For scale functions in visualizations.
-   **svglite** (\>= 2.1.3): For creating high-quality vector graphics (SVG).
-   **tibble** (\>= 3.2.1): For creating tidy data frames.
-   **tidyr** (\>= 1.3.1): For tidying data.
-   **zip** (\>= 2.3.1): For compressing and combining files into zip archives.

### Optional Dependencies

These packages are optional and are only needed for specific functionality:

-   **edgeR** (\>= 4.0.16): For preprocessing RNA-seq data in the `preprocess_rna_seq_data()` function.
-   **clusterProfiler** (\>= 4.10.1): For the `run_gsea()` function (gene set enrichment analysis).
-   **rstudioapi** (\>= 0.16.0): For the `open_tutorial()` and `open_template()` functions.

### R Version

-   Recommended: R 4.3.3 or higher

## 📚 Further Reading

For those interested in gaining a deeper understanding of the methodologies used in the `SplineOmics` package, here are some recommended publications:

-   **Splines**: To learn more about splines, you can refer to this [review](https://doi.org/10.1186/s12874-019-0666-3).

-   **limma**: To read about the `limma` R package, you can refer to this [publication](https://doi.org/10.1093/nar/gkv007).

-   **PCA**: To learn more about PCA, download and read this [document](https://github.com/csbg/SplineOmics/raw/main/docs/Points_of_Significance_PCA.pdf).

-   **Hierarchical clustering**: To get information about hierarchical clustering, you can refer to this [web article](https://towardsdatascience.com/understanding-the-concept-of-hierarchical-clustering-technique-c6e8243758ec).

## ❓ Getting Help

If you encounter a bug or have a suggestion for improving the `SplineOmics` package, we encourage you to [open an issue](https://github.com/csbg/SplineOmics/issues) on our GitHub repository. Before opening a new issue, please check to see if your question or bug has already been reported by another user. This helps avoid duplicate reports and ensures that we can address problems efficiently.

For more detailed questions, discussions, or contributions regarding the package’s use and development, please refer to the [GitHub Discussions](https://github.com/csbg/SplineOmics/discussions) page for `SplineOmics`.

## 🤝 Contributing

We welcome contributions to the `SplineOmics` package! Whether you’re interested in fixing bugs, adding new features, or improving documentation, your help is greatly appreciated.

Here’s how you can contribute:

1.  **Report a Bug or Request a Feature:** If you encounter a bug or have an idea for a new feature, please [open an issue](https://github.com/csbg/SplineOmics/issues) on our GitHub repository. Before opening a new issue, check to see if the issue has already been reported or the feature requested by another user.

2.  **Submit a Pull Request:** If you’ve developed a bug fix or a new feature that you’d like to share, submit a pull request.

3.  **Improve Documentation:** Good documentation is crucial for any project. If you notice missing or incorrect documentation, please feel free to contribute.

Please adhere to our [Code of Conduct](CODE_OF_CONDUCT.md) in all your interactions with the project.

Thank you for considering contributing to `SplineOmics`. Your efforts are what make the open-source community a fantastic place to learn, inspire, and create.

## 💬 Feedback

We appreciate your feedback! Besides raising issues, you can provide feedback in the following ways:

-   **Direct Email**: Send your feedback directly to [Thomas Rauter](mailto:thomas.rauter@plus.ac.at).

-   **Anonymous Feedback**: Use [this Google Form](https://forms.gle/jocMXSxLf3GrGBdT9) to provide anonymous feedback by answering questions.

Your feedback helps us improve the project and address any issues you may encounter.

## 📜 License

This package is licensed under the MIT License: [LICENSE](./LICENSE)

© 2024 Thomas Rauter. All rights reserved.

## 🎓 Citation

The `SplineOmics` package is currently not published in a peer-reviewed scientific journal or similar outlet. However, if this package helped you in your work, consider citing this GitHub repository.

To cite this package, you can use the citation information provided in the [`CITATION.cff`](./CITATION.cff) file.

You can also generate a citation in various formats using the `CITATION.cff` file by visiting the top right of this repo and clicking on the “Cite this repository” button.

Also, if you like the package, consider giving the GitHub repository a star. Your support helps us in the continued development and improvement of `SplineOmics`. Thank you for using our package!

## 🌟 Contributors

-   [Thomas-Rauter](https://github.com/Thomas-Rauter) - 🚀 Wrote the package, developed the approach together with VSchaepertoens under guidance from nfortelny and skafdasschaf.
-   [nfortelny](https://github.com/nfortelny) - 🧠 Principal Investigator, provided guidance and support for the overall approach.
-   [skafdasschaf](https://github.com/skafdasschaf) - 🔧 Helped reviewing code, delivered improvement suggestions and scientific guidance to develop the approach.
-   [VSchaepertoens](https://github.com/VSchaepertoens) - ✨ Developed one internal plotting function, as well as some code for the exploratory data analysis plots, and the overall approach together with Thomas-Rauter.

## 🙏 Acknowledgements

This work was carried out in the context of the DigiTherapeutX project, which was funded by the Austrian Science Fund (FWF). The research was conducted under the supervision of Prof. Nikolaus Fortelny, who leads the Computational Systems Biology working group at the Paris Lodron University of Salzburg, Austria. You can find more information about Prof. Fortelny's research group [here](https://www.plus.ac.at/biowissenschaften/der-fachbereich/arbeitsgruppen/fortelny/).