Table of contents

• BSXplorer
  • How to cite
  • Installation
  • Usage
  • API usage
    • Basic usage
    • Methylation pattern clusterisation
    • Chromosome methylation levels
    • Gene body methylation
    • Different organisms analysis
    • Enrichment of DMRs
    • Other functionality
  • Console usage
  • What's new

BSXplorer

Analytical framework for BS-seq data comparison and visualization. BSXplorer facilitates efficient methylation data mining, contrasting and visualization, making it an easy-to-use package that is highly useful for epigenetic research.

For Python API reference manual and tutorials visit: https://shitohana.github.io/BSXplorer.

How to cite

## How to cite If you use our package in your research, please consider citing our paper. Yuditskiy, K., Bezdvornykh, I., Kazantseva, A. et al. BSXplorer: analytical framework for exploratory analysis of BS-seq data. BMC Bioinformatics 25, 96 (2024). https://doi.org/10.1186/s12859-024-05722-9

Installation

To install latest stable version:

commandline pip install bsxplorer

If you want to install the prerelease version (dev branch):

commandline pip install pip install git+https://github.com/shitohana/BSXplorer.git@dev

Usage

In this project our aim was to create a both powerful and flexible tool to facilitate exploratory data analysis of BS-Seq data obtained in non-model organisms (BSXplorer works for model organisms as well). That's why BSXplorer is implemented as a Python package. Modular structure of BSXplorer together with easy to use and configurable API makes it a highly integratable and scalable package for a wide range of applications in bioinformatical projects.

Even though BSXplorer is available as console application, to fully utilize its potential consider using it as a python package. Detailed documentation can be found here.

API usage

python import bsxplorer as bsx

Basic usage

The main objects in BSXplorer are the Genome and Metagene, MetageneFiles classes. Genome class is used for reading and filtering genomic annotation data.

python genome = bsx.Genome.from_gff("path/to/annotation.gff")

Even though here genome was created with .from_gff constructor, to read custom annotation format (TSV file), use .from_custom and specify column indexes (0-based).

Once we have read annotation file, methylation report can be processed via Metagene class (or MetageneFiles for multiple reports).

python metagene = bsx.Metagene.from_bismark( "path/to/report.txt", genome=genome.gene_body(min_length=0, flank_length=2000), up_windows=100, body_windows=200, down_windows=100 )

Here we have read methylation report file. Methylation data has been read only for gene bodies (genome.gene_body(min_length=0, flank_length=2000)) with 200 windows resolution for gene body (body_windows=200) and 100 for flanking regions (up_windows=100, down_windows=100).

Now we can generate visualiztions.

python filtered = metagene.filter(context="CG") filtered.line_plot().draw_mpl() filtered.heat_map().draw_mpl()

BSXplorer can generate plots with two plotting libraries: matplotlib and Plotly. _mpl in methods names stands for matplotlib and _plotly for Plotly.

Clusterisation

BSXplorer allows for discovery of gene modules characterised with similar methylation patterns.

Once the data was filtered based on methylation context and strand, one can use the .cluster() method. The resulting object contains an ordered list of clustered genes and their visualisation in a form of a heatmap.

Chromosome methylation levels

BSXplorer allows a user to visualize the overall methylation levels of chromosomes using the corresponding ChrLevels object:

python levels = bsx.ChrLevels.from_bismark("path/to/report.txt", chr_min_length=10**6, window_length=10**6) levels.draw_mpl(smooth=5)

In a way that is similar to the Metagene method, the methylation data can be subjected to filtering to selectively display a methylation context that is of interest.

python levels.filter(context="CG").draw_mpl(smooth=5)

Gene body methylation

BSXplorer allows for the categorization of regions based on their methylation level and density. This is done by assuming that cytosine methylation levels follow a binomial distribution, as explained in Takuno and Gaut's research (please refer to [1, 2] https://doi.org/10.1073/pnas.1215380110 for details). The genes are then divided into three categories, BM (body-methylated), IM (intermediately-methylated) and UM (under-methylated), based on their methylation levels in the CG context using the following formula.

$$ CG1-P_{CG} $$

$$ P{CG}\le CG<1-P{CG};\ \ CHG/CHH>1-P_{CG} $$

$$ CG/CHG/CHH>1-P_{CG} $$

The same rationale may be applied to other methylation contexts, as BSXplorer can produce $P{CHG}$ and $P{CHH}$ for CHG sites and CHH sites, respectively.

[1] Takuno S, Gaut BS. Body-Methylated Genes in Arabidopsis thaliana Are Functionally Important and Evolve Slowly. Mol Biol Evol. 2012;29:219–27.

[2] Takuno S, Gaut BS. Gene body methylation is conserved between plant orthologs and is of evolutionary consequence. Proc Natl Acad Sci. 2013;110:1797–802.

```python

Calculate pvalue for cytosine methylation via binomial test

binomdata = bsx.BinomialData.fromreport( "path/to/report.txt", report_type="bismark" )

Created binomial data object can now be used to calculate pvalues

for methylation of genomic regions

regionstats = binomdata.regionpvalue(genome.genebody(), methylation_pvalue=.01)

.categorise method returns tuple of three DataFrames

for BM, IM and UM genes respectively

bm, im, um = regionstats.categorise(context="CG", pvalue=.05)

Now we can create MetageneFiles object to visualize methylation pattern

of categorised groups

cat_metagene = bsx.MetageneFiles([ metagene.filter(context="CG", genome=bm), metagene.filter(context="CG", genome=im), metagene.filter(context="CG", genome=um), ], labels=["BM", "IM", "UM"])

And plot it

ticklabels = ["-2000kb", "TSS", "", "TES", "+2000kb"] catmetagene.lineplot().drawmpl(ticklabels=ticklabels) ```

Different organisms analysis

Start with import of genome annotation data for species of interest.

python arath_genes = bsxplorer.Genome.from_gff("arath_genome.gff").gene_body(min_length=0) bradi_genes = bsxplorer.Genome.from_gff("bradi_genome.gff").gene_body(min_length=0) mouse_genes = bsxplorer.Genome.from_gff("musmu_genome.gff").gene_body(min_length=0)

Next, read in cytosine reports for each sample separately:

```python windowkwargs = dict(upwindows=200, bodywindows=400, downwindows=200)

arathmetagene = bsx.Metagene.frombismark("arathexample.txt", arathgenes, *windowkwargs) bradimetagene = bsx.Metagene.frombismark("bradiexample.txt", bradi_genes, *windowkwargs) musmumetagene = bsx.Metagene.frombismark("musmuexample.txt", mousegenes, **windowkwargs) ```

To perform comparative analysis, initialize the bsxplorer.MetageneFiles class using metagene data in a vector format, where labels for every organism are provided explicitly.

Next, apply methylation context and strand filters to the input files:

python filtered = files.filter("CG", "+")

Then, a compendium of line plots to guide a comparative analyses of methylation patterns in different species is constructed:

python filtered.line_plot(smooth=50).draw_mpl()

The line plot representation may be further supplemented by a heatmap:

python filtered.heat_map(100, 100).draw_mpl()

To examine and highlight differences in methylation patterns between different organisms, summary statistics is made available in a graphical format.

python filtered.box_plot(violin=True).draw_mpl() filtered.box_plot().draw_mpl()

Enrichment of DMRs

BSXplorer offers functionality to align one set of regions over another. Regions can be read either with :class:Genome or initialized directly with polars functionality <https://docs.pola.rs/api/python/stable/reference/api/polars.read_csv.html>_ (DataFrame need to have chr, start and end columns).

To align regions (e.g. define DMR position relative to genes) or perform the enrichment of regions at these genomic features against the genome background use :class:Enrichment.

```python

If you want to perform an ENRICHMENT, and not only plot

the density of metagene coverage, you NEED to use .raw() method

for genome DataFrame.

genes = bsx.Genome.fromgff("path/to/annot.gff").raw() dmr = bsx.Genome.fromcustom( "path/to/dmr.txt", chrcol=0, # Theese columns indexes are configurable startcol=1, end_col=2 ).all()

enrichment = bsx.Enrichment(dmr, genes, flank_length=2000).enrich() ```

Enrichment.enrich returns EnrichmentResult, which stores enrichment statistics and coordinates of regions which have aligned with genomic features. The metagene coverage with regions can be plotted via EnrichmentResult.plot_density_mpl method.

python fig = enrichment.plot_density_mpl( tick_labels=["-2000bp", "TSS", "Gene body", "TES" "+2000bp"], )

Enrichment statistics can be accessed with EnrichmentResult.enrich_stats or plotted with EnrichmentResult.plot_enrich_mpl

python enrichment.plot_enrich_mpl()

Other functionality

For other functionality, such as methylation reports conversion and BAM conversion and statistics please refer to the documentation.

Console usage

BSXplorer can be used in a console mode for generating complex HTML-reports (see example here) and running many analysis at once or converting BAM to methylation report. For detailed commands description and examples, please refer to the documentation.

What's new

Since publication we have released Version 1.1.0.

Major changes

• Added new classes for Unified reading of methylation reports (UniversalReader, UniversalReplicatesReader). Now any supported report type can be converted into another.

• Added support for processing BAM files (BAMReader). BAM files can be either converted to methylation report (faster than with native methods), or methylation statistics, such as methylation entropy, epipolymorphism or PDR can be calculated.

• Added method for aligning one set of regions along another (e.g. DMR along genes) – Enrichment. Regions can not only be aligned, but the coverage of the metagene by DMRs can be visualized.

Other improvements

• Any plot data now can be retrieved by corresponding method.
• Fixes to the plotting API.
• Fixes to Category report.
• Added console command for processing BAM files.