🧬 methurator

Methurator is a Python package designed to estimate CpGs saturation for DNA methylation sequencing data.

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📑 Table of Contents

• 1. Dependencies and Notes
• 2. Installation
• 3. Quick Start
  • Option A: Chao Estimator
  • Option B: Downsample
  • Step 3 — Plot the sequencing saturation curve
• 4. Command Reference
  • gt-estimator command
  • downsample command
  • plot command
• 5. Example Workflow
• 6. How do we compute the sequencing saturation?

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1. Dependencies and Notes

• methurator uses SAMtools and MethylDackel internally for BAM subsampling, thus they need to be installed.
• When --genome is provided, the corresponding FASTA file will be automatically fetched and cached.
• Temporary intermediate files are deleted by default unless --keep-temporary-files is specified.

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2. Installation

You can install methurator in several ways:

Option 1: Install via pip

pip install methurator

Option 2: Install via BioConda

conda create -n methurator_env conda::methurator
conda activate methurator_env

Option 3: Use the BioContainer

docker pull quay.io/biocontainers/methurator:2.1.1--pyhdfd78af_0
docker run quay.io/biocontainers/methurator:2.1.1--pyhdfd78af_0 methurator -h

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3. Quick Start

Option A: Chao Estimator (best practise)

The gt-estimator command estimates CpGs sequencing saturation at higher depth than the observed one. This is the recommended approach for extrapolation analysis.

methurator gt-estimator --fasta tests/data/genome.fa tests/data/Ecoli.csorted.bam

This command generates:

• Summary YAML file (methurator_summary.yml) — Contains metadata, model parameters, and extrapolation results with:
  • Extrapolation factor (t) values from 0 to --t-max (default: 10.0)
  • Boolean indicating interpolated (t ≤ 1) vs extrapolated (t > 1) data
  • Total CpGs predicted at each t value
  • Theoretical asymptote (maximum CpGs, computed at t = 1000)
  • Number of reads observed at full sequencing depth (t = 1)
  • Confidence intervals (if --compute_ci is enabled)

Option B: Downsample

The downsample command performs BAM downsampling according to specified percentages and coverage levels:

methurator downsample --fasta tests/data/genome.fa tests/data/Ecoli.csorted.bam

This command generates:

• CpG summary — number of unique CpGs detected in each downsampled BAM
• Reads summary — number of reads in each downsampled BAM
• Summary YAML — consolidated file with all data and run metadata

Plot the sequencing saturation curve

Use the plot command to visualize the results, including the asymptote line and the number of reads at each t:

methurator plot --summary output/methurator_summary.yml

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4. Command Reference

gt-estimator command

Argument	Description	Default
BAM (positional)	Path to a single .bam file or to multiple ones (e.g. files/*.bam).	—
--outdir, -o	Output directory.	./output
--fasta	Path to the reference genome FASTA file. If not provided, it will be automatically downloaded based on --genome.	—
--genome	Genome used for alignment. Available: hg19, hg38, GRCh37, GRCh38, mm10, mm39.	—
--minimum-coverage, -mc	Minimum CpG coverage to consider. Can be a single integer or a list (e.g. 1,3,5).	1
--t-step	Step size for extrapolation factor (t) predictions.	0.05
--t-max	Maximum extrapolation factor (t) value.	10.0
--compute_ci	Compute confidence intervals using bootstrap replicates.	False
--bootstrap-replicates, -b	Number of bootstrap replicates for CI computation.	30
--conf	Confidence level for bootstrap intervals.	0.95
--mu	Initial mu parameter for negative binomial distribution in EM algorithm.	0.5
--size	Initial size parameter for negative binomial distribution in EM algorithm.	1.0
--mt	Constraint for rational function approximations.	20
--rrbs	If set to True, MethylDackel will use the RRBS flag (--keepDupes).	False
--threads, -@	Number of threads to use.	Available threads - 2
--keep-temporary-files, -k	Keep temporary files after analysis.	False
--verbose	Enable verbose logging.	False
--help , -h	Print the help message and exit.
--version	Print the package version.

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downsample command

Option	Description	Default
BAM (positional)	Path to a single .bam file or to multiple ones (e.g. files/*.bam).	—
--outdir, -o	Output directory.	./output
--fasta	Path to the reference genome FASTA file. If not provided, it will be automatically downloaded based on --genome.	—
--genome	Genome used for alignment. Available options: hg19, hg38, GRCh37, GRCh38, mm10, mm39.	—
--downsampling-percentages, -ds	Comma-separated list of downsampling percentages between 0 and 1 (exclusive).	0.1,0.2,0.4,0.6,0.8
--minimum-coverage, -mc	Minimum CpG coverage to consider for saturation. Can be a single integer or a list (e.g. 1,3,5).	3
--rrbs	If set, MethylDackel extract will consider the RRBS nature of the data by adding the --keepDupes flag.	False
--threads, -@	Number of threads to use during downsampling.	All available threads
--keep-temporary-files	If set, temporary files will be kept after analysis.	False
--verbose	Enable verbose logging.	False
--help, -h	Print the help message and exit.	—
--version	Print the package version.	—

plot command

Argument	Description	Default
--summary, -s	Path to the YML summary file.
--outdir, -o	Output directory.	./output
--verbose	Enable verbose logging.	False
--help , -h	Print the help message and exit.
--version	Print the package version.

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5. Example Workflow

Using Chao Estimator (Recommended)

# Run Chao estimator on BAM file
methurator gt-estimator --genome hg19 my_sample.bam --config_ci

# Generate plots from the results
methurator plot --summary output/methurator_summary.yml

Example plot preview (also available as interactive html file here):

Using Downsample

# Downsample BAM file
methurator downsample --genome hg19 my_sample.bam

# Generate plots from the results
methurator plot --summary output/methurator_summary.yml

The output plots will be saved in output/plots/ as interactive HTML files showing the CpG predictions, the asymptote (theoretical maximum CpGs at t = 1000), the number of reads at each t, and confidence intervals (if enabled). The asymptote is now also used to compute the saturation values.

Example plot preview (also available as interactive html file here):

6. How do we compute the sequencing saturation?

Chao Estimator approach (best practise)

methurator gt-estimator uses an approach developed in 2018 by Chao Deng et al and further implemented in preseqR. This approach builds on the theoretical nonparametric empirical Bayes foundation of Good and Toulmin (1956), to model sequencing saturation and extrapolate to higher sequencing depths. The model implemented in preseqR was mirrored here and tailored toward sequencing saturation application. The workflow consists of the following steps:

1. Extracts CpGs from BAM files using MethylDackel
2. Fits the model implemented by Chao Deng et al taking in input the observed CpG counts
3. Predicts future CpG discovery using rational function approximations
4. Quantifies confidence intervals through bootstrap resampling (if enabled)

The extrapolation factor (t) represents the ratio of hypothetical total reads to actual observed reads. Values of t ≤ 1 correspond to interpolation (between observed data points), while t > 1 represents extrapolation (prediction beyond observed depth).

For a given coverage level:

• At t = 1: prediction matches observed CpGs, and the number of reads at full sequencing depth is reported in the summary file
• As t increases: predictions approach the theoretical asymptote (maximum CpGs at t = 1000, shown in the plot and used for saturation calculation)

Downsample approach

To calculate the sequencing saturation of an DNAm sample when using the downsample command, we adopt the following strategy. For each sample, we downsample it according to 4 different percentages (default: 0.1,0.2,0.4,0.6,0.8). Then, we compute the number of unique CpGs covered by at least 3 reads and the number of reads at each downsampling percentage.

We then fit the following curve using the scipy.optimize.curve_fit function:

$$ y = \beta_0 \cdot \arctan(\beta_1 \cdot x) $$

We chose the arctangent function because it exhibits an asymptotic growth similar to sequencing saturation. For large values of $\text{x}$ (as $\text{x} \to \infty$), the asymptote corresponds to the theoretical maximum number of unique CpGs covered by at least 3 reads and can be computed as:

$$ \text{asymptote} = \beta_0 \cdot \frac{\pi}{2} $$

Finally, the sequencing saturation value can be calculated as following:

$$ \text{Saturation} = \frac{\text{Number of unique CpGs (≥3 counts)}}{\text{Asymptote}} $$

This approach allows estimation of the theoretical maximum number of CpGs that can be detected given an infinite sequencing depth, and quantifies how close the sample is to reaching sequencing saturation.