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https://github.com/smaegol/nanotail
R package for visualization and exploratory analysis of Oxford Nanopore direct RNA seq based polyA predictions
https://github.com/smaegol/nanotail
bioinformatics nanopolish nanopore nanopore-sequencing polya polya-tail rna rna-sequencing shiny
Last synced: 10 days ago
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R package for visualization and exploratory analysis of Oxford Nanopore direct RNA seq based polyA predictions
- Host: GitHub
- URL: https://github.com/smaegol/nanotail
- Owner: smaegol
- License: gpl-3.0
- Created: 2019-03-21T13:39:43.000Z (almost 6 years ago)
- Default Branch: master
- Last Pushed: 2024-06-25T19:04:22.000Z (6 months ago)
- Last Synced: 2024-06-25T20:53:58.593Z (6 months ago)
- Topics: bioinformatics, nanopolish, nanopore, nanopore-sequencing, polya, polya-tail, rna, rna-sequencing, shiny
- Language: R
- Homepage:
- Size: 20.9 MB
- Stars: 8
- Watchers: 3
- Forks: 5
- Open Issues: 2
-
Metadata Files:
- Readme: README.md
- License: LICENSE
Awesome Lists containing this project
README
# nanotail
[](https://travis-ci.org/smaegol/nanotail)
[](https://codecov.io/gh/smaegol/nanotail)
[](https://zenodo.org/badge/latestdoi/176952175)
[](https://www.gnu.org/licenses/gpl-3.0.en.html)
[
The goal of **NanoTail** is to provide a set of functions to manipulate and analyze data coming from polyA lengths estimations done using Oxford Nanopore Direct RNA sequencing and Nanopolish software. Existing solutions, like [Pipeline for testing shifts in poly(A) tail lengths estimated by nanopolish](https://github.com/nanoporetech/pipeline-polya-diff/) are, in our opinion, not sufficient for in-depth analysis of such data.
The software is still in the development phase so all suggestions are welcome. Please also expect the code to be changed frequently, so use it with caution.
## Installation
### Prerequisities
As `asserrtive` package was [removed from CRAN](https://cran.r-project.org/web/packages/assertive/index.html) it is currently impossible to install nanotail without prior manual installation of assertive package. We are working on exchanging the `assertive` package with its equivalent. However, for now, it is required to install all assertive packages manually:
```r
install.packages("devtools")
devtools::install_bitbucket("richierocks/assertive.properties") # install this one first as other packages depend on it
devtools::install_bitbucket(c("richierocks/assertive.files", "richierocks/assertive.strings", "richierocks/assertive.numbers", "richierocks/assertive.matrices", "richierocks/assertive.sets", "richierocks/assertive.strings", "richierocks/assertive.models", "richierocks/assertive.reflection", "richierocks/assertive.types", "richierocks/assertive.datetimes", "richierocks/assertive.data", "richierocks/assertive.data.uk", "richierocks/assertive.data.us", "richierocks/assertive.code"))
devtools::install_bitbucket("richierocks/assertive.properties") # install this one first as other packages depend on it
devtools::install_bitbucket("richierocks/assertive")
```
Now you can install the developmental version of Nanotail with
``` r
devtools::install_github('smaegol/nanotail')
library(nanotail)
```
## Input data
NanoTail needs output from [nanopolish](https://github.com/jts/nanopolish) polya to work. It can read a single output file with `read_polya_single`:
``` r
path <- "/location/of/nanopolish/output"
polya_data <- read_polya_single(path)
```
It can also read multiple samples at once and associate any metadata with them.
Let's assume we have performed an experiment, targeting one of the polyA polymerases. 2 replicates were sequenced for control samples, and 2 replicates sequenced for samples with mutant PAP, therefore after all analysis we have 4 files with [nanopolish](https://github.com/jts/nanopolish) polya output. To read all of them at once, we can use command `read_polya_multiple` and associate metadata using samples_table data.frame:
``` r
samples_table <- data.frame(polya_path = c(path1,path2,path3,path4),
sample_name =c("wt1","mu1","wt2","mut2"),
group = c("wt","mut","wt","mut"))
polya_data_multiple <- read_polya_multiple(samples_table)
```
To obtain nanopolish predictions one can use [Pipeline for calling poly(A) tail lengths from nanopore direct RNA data using nanopolish](https://github.com/nanoporetech/pipeline-polya-ng)
## Shiny App
Once data are imported they can be processed in the R environment using NanoTail functions described below or, more convenient, the interactive Shiny app can be launched, allowing for easy exploration of obtained data. To launch the app for the data imported above:
``` r
nanoTailApp(polya_table = polya_data_multiple)
```
## Nanopolish output QC
To get overall information about the output of NanoPolish polya analysis, please use `get_nanopolish_processing_info()` function. Obtained summary can be plotted using `plot_nanopolish_qc()`. Summary of the analysis is also shown in the *QC info* tab of the Shiny App.
## Global distribution of polyA lengths
Global distribution of polyA tails lengths can be plotted with `plot_polya_distribution()` function, which produces the density plot, allowing for comparison of the distribution of polyA lengths between samples. The same plot can be seen in the *Global polya distribution* tab of the Shiny App.
## Statistical analysis of polyA predictions
NanoTail is intended to analyze differential adenylation. For this purpose 3 statistical tests can be employed, allowing or comparison of polyA lengths of individual transcripts between selected conditions:
* Wilcoxon rank-sum test ([Mann-Whitney U-test](https://en.wikipedia.org/wiki/Mann%E2%80%93Whitney_U_test))
* [Kolmogorov-Smirnov test](https://en.wikipedia.org/wiki/Kolmogorov%E2%80%93Smirnov_test) of the equality of distributions
* [generalized linear model](https://en.wikipedia.org/wiki/Generalized_linear_model) with log(polya_length) as the response, and post-hoc Tukey test
Differential adenylation analysis can be performed with the `calculate_polya_stats` function, or within the *Differential adenylation* tab in the Shiny App.
## Differential expression analysis
NanoTail provides also the possibility of very basic differential expression testing, using binomTest from the [edgeR](https://bioconductor.org/packages/release/bioc/html/edgeR.html) package. This functionality is still in the development and may not work as expected. To calculate differential expression please use `calculate_diff_exp_binom()` function or use Shiny App.
## Citation
Please cite NanoTail as:
Krawczyk PS et al., NanoTail - R package for exploratory analysis of Nanopore Direct RNA based polyA lengths estimations
Preprint in the preparation.
## TBD & plans
* Import of polya predictions from software other then NanoPolish (poreplex,tailfindr,?)
* Analysis of predictions based on genome-mapping (now only transcriptome-mapping is supported)
* Annotation of results and enrichment analysis
* Squiggle visualization of polyA tails
## Support
Any issues connected with the NanoTail should be addressed to Pawel Krawczyk (p.krawczyk (at) ibb.waw.pl).