# RifiComparative ## About RifiComparative is a workflow designed for comparative analysis of data generated by the Rifi framework (https://github.com/CyanolabFreiburg/rifi). It addresses the challenge of comparing outputs from two different conditions of the same organism, where the transcriptome regulation leads to variations in the Rifi outputs. These variations result in segments with different lengths, positions, and events, making a direct comparison between the two conditions difficult (Figure 1). To overcome this issue, we have developed a strategy (Figure 2). The data from both conditions are combined, and a segmentation approach is applied to determine the half-life and mRNA at time 0 segments for each condition. The absolute value of the half-life difference at the probe level serves as input for segmenting the half-life, while the fold-change of mRNA at time 0 is used for segmenting the intensity. Unlike Rifi, no hierarchical segmentation is employed (Figure 3). Prior to segmentation, a subset of the data is trained to optimize the penalty parameters and ensure the best segmentation results. After segmentation, the fragments are subjected to statistical testing, and a p-value is assigned. To enable a meaningful comparison, the fragments from the half-life and mRNA at time 0 segments are adjusted to align with each other, resulting in fragments of equal length for both parameters (Figure 4). Each fragment corresponds to a specific annotation on the genome. The package provides the capability to plot the half-life and intensity fragments, transcription units (TUs) from each condition, termination events, iTSS_II events, and genome annotations (Figure 5). Furthermore, a series of additional plots are included to facilitate the analysis. For instance, Figure 6 and 7 display decay rate versus synthesis rate, with the option to highlight or label points of interest using the segment annotation. RifiComparative offers a comprehensive workflow for comparative analysis of Rifi data, allowing for improved comparisons between different conditions of the same organism. The package provides various visualization tools to aid in the interpretation and exploration of the results. <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/rifiComparative_workflow.png"/> </p> _{<b>Figure 1:</b> rifi Comparative workflow. Four major steps are depicted. 1) Gathering the data from two conditions, 2) penalties set, 3) segmentation, 4) adjusting the fragments to each other, 5) genome visualization together with the fragment.} <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/Half-life_Fragments_two_conditions.png"/> </p> _{<b>Figure 2:</b> Segments output of Rifi. The segments are very different making the comparison nearly impossible.} <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/Half-life_difference.png"/> </p> _{<b> Figure 3:</b> Half-life difference. Difference between Half-life from both conditions is calculated by bin or probe (distance = abs(diff(half-life(cdt 1)(Pi) - half-life(cdt 2)(Pi)))). cdt = condition and Pi = position i.} <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/adjusting_fragments.png"/> </p> _{<b> Figure 4:</b> Adjusting half-life and intensity to each other. The adjustment was applied only if at least one fragment has a significant p_value. Numbers are just fragments indicator.} <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/Decay_rate_vs_Synthesis_rate.png" alt="drawing" width="400"/> </p> _{<b> Figure 5:</b> Plot changes in RNA decay rates (log fold, x-axis) versus the changes in RNA synthesis rates (log fold, y-axis) in the condition 1 versus condition 2. The black lines horizontal, vertical and diagonal are the median of synthesis_rate, decay_rate and mRNA at time 0 respectively. Dashed gray lines indicate 0.5-fold changes from 0 (gray lines) referring to unchanged fold. The points highlighted with yellow color show a decay rate \>= .5 and synthesis rate \<= -.5.} <br/> <p align="center"> <img src="https://github.com/CyanolabFreiburg/rifiComparative/blob/main/vignettes/heatscatter_Decay_rate_vs_Synthesis_rate.png" alt="drawing" width="600"/> </p> _{<b> Figure 6:</b> Plot of the changes in RNA decay rates (log fold, x-axis) versus the changes in RNA synthesis rates (log fold, y-axis) in the condition 1 versus condition 2. The coloring indicates the local point density.} # Installation ### Dependencies RifiComparative package only available for Unix systems and has the following dependencies. Make sure the requirements are satisfied by your system. [devtools](https://rdocumentation.org/packages/devtools/versions/2.4.3)(\>= 2.4.3) [roxygen2](https://cran.r-project.org/web/packages/roxygen2/index.html)(\>= 7.1.2) [SummarizedExperiment](https://bioconductor.org/packages/release/bioc/html/SummarizedExperiment.html)(\>= 1.24.0) [dplyr](https://rdocumentation.org/packages/dplyr/versions/0.7.8) (\>= 0.7.8) [LSD](https://www.rdocumentation.org/packages/ScottKnott/versions/1.3-0/) (\>= 1.3.0) [ggplot2](https://ggplot2.tidyverse.org/) (\>= 3.3.5) [writexl](https://www.rdocumentation.org/packages/writexl/versions/1.4.0/) (\>= 1.4.0) [egg](https://www.rdocumentation.org/packages/scales/versions/0.4.1) (\>= 0.4.1) [data.table](https://www.rdocumentation.org/packages/msSPChelpR/versions/0.8.7/) (\>= 0.8.7) [ggrepel](https://www.rdocumentation.org/packages/ggrepel/versions/0.9.1/) (\>= 0.9.1) [stats](https://rdocumentation.org/packages/stats/versions/3.6.2) (\>= 3.6.2) [grid](https://www.rdocumentation.org/packages/graphics/versions/3.6.2/topics/grid) (\>= 3.6.2) [rtracklayer](https://www.rdocumentation.org/packages/rtracklayer/versions/1.32.1) (\>= 1.32.1) [reshape2](https://rdocumentation.org/packages/reshape2/versions/1.4.4) (\>= 1.4.4) [utils](https://rdocumentation.org/packages/utils/versions/3.6.2) (\>= 3.6.2) [DTA](https://rdocumentation.org/packages/DTA/versions/2.18.0)(\>= 2.18.0) [cowplot](https://rdocumentation.org/packages/cowplot/versions/1.1.1)(\>= 1.1.1) [doMC](https://cran.r-project.org/web/packages/doMC/index.html) (\>= 1.3.7) [parallel](https://rdocumentation.org/packages/parallel/versions/3.6.2) (\>= 3.6.2) [graphics](https://rdocumentation.org/packages/graphics/versions/3.6.2) (\>= 3.6.2) [stats](https://rdocumentation.org/packages/stats/versions/3.6.2) (\>= 3.6.2) [stringr](https://www.rdocumentation.org/packages/stringr/versions/1.4.0) (\>= 1.4.0) [grDevices](https://rdocumentation.org/packages/grDevices/versions/3.6.2) (\>= 3.6.2) [tibble](https://rdocumentation.org/packages/tibble/versions/3.1.6) (\>= 3.1.6) [methods](https://rdocumentation.org/packages/methods/versions/3.6.2) (\>= 3.6.2) ### To install from Bioconductor, use the following code: ``` html if (!requireNamespace("BiocManager", quietly=TRUE)) install.packages("BiocManager") BiocManager::install("rifiComparative") ``` ### To install directly from github: ``` html install_github('rifiComparative') ``` # Troubleshooting contact [lyoussar\@gmail.com](mailto:lyoussar@gmail.com) or create an issue