METHPLOTLIB

This script generates a browser view on a window using data from
i) nanopolish, either as methylation calls or methylation frequencies (as processed by calculate_methylation_frequency.py). The methylation calls can additionally be phased using scripts/annotate_calls_by_phase.py and scripts/split_calls_by_phase.py
ii) nanocompore
iii) in ont-cram format with MM/ML tags according to the SAM specifications
iv) in bedgraph format, with 4 columns (chromosome, start, end, value) or the 5 columns written by modkit pileup --bedgraph
v) in bedMethyl format, as written by modkit pileup (18 columns, with or without --only-tabs) or by modbam2bed --extended (14 columns). Files containing multiple modifications (e.g. 5mC and 5hmC) do not have to be split up: each modification gets its own track, and --mods can be used to restrict to some of them

INSTALLATION

Creating a new conda environment:
conda create -n methplotlib methplotlib

Or using pip:
pip install methplotlib

USAGE

methplotlib [-h] [-v] -m METHYLATION [METHYLATION ...] -n NAMES
                   [NAMES ...] -w WINDOW [-g GTF] [-b BED] [-f FASTA]
                   [--simplify] [--split] [--static STATIC] [--smooth SMOOTH]
                   [--dotsize DOTSIZE] [--example] [-o OUTFILE] [-q QCFILE]

plotting nanopolish methylation calls or frequency

optional arguments:
  -h, --help            show this help message and exit
  -v, --version         Print version and exit.
  -m, --methylation METHYLATION [METHYLATION ...]
                        data in nanopolish, nanocompore, ont-cram or bedgraph
                        format
  -n, --names NAMES [NAMES ...]
                        names of datasets in --methylation
  -w, --window WINDOW   window (region) to which the visualisation has to be restricted
  -g, --gtf GTF         add annotation based on a gtf file
  -b, --bed BED         add annotation based on a bed file
  -f, --fasta FASTA     required when --window is an entire chromosome, contig or transcript
  --simplify            simplify annotation track to show genes rather than transcripts
  --split               split, rather than overlay the methylation tracks
  --static              Make a static image of the browser window (filename)
  --binary              Make the nanopolish plot ignorning log likelihood nuances
  --smooth              Rolling window size for averaging frequency values (int)
  --dotsize             Control the size of dots in the per read plots (int)
  --example             Show example command and exit.
  -o, --outfile OUTFILE File to write results to. Default:
                        methylation_browser_{chr}_{start}_{end}.html. Use
                        {region} as a shorthand for {chr}_{start}_{end} in the
                        filename. Missing paths will be created.
  -q, --qcfile QCFILE   File to write the qc report to. Default: The path in
                        outfile prefixed with qc_, default is qc_report_methyl
                        ation_browser_{chr}_{start}_{end}.html. Use {region}
                        as a shorthand for {chr}_{start}_{end} in the
                        filename. Missing paths will be created.

Snakemake workflow

For streamlining nanopolish a Snakefile is included (using snakemake). The workflow uses a config file, of which an example is in this repository.

Example data

The examples folder contains calls and frequencies for the human ACTB gene from PromethION sequencing of NA19240. An example command is available.

Companion scripts

The scripts folder contains scripts for phasing modification calls in haplotypes based on WhatsHap phasing, allele specific modification testing for phased data and differential modification testing across subjects.

TO DO - CONTRIBUTIONS WELCOME

• Outlier detection (in windows) across samples