bygul 3.2.0

Amplicon read simualtor

Bygul: Amplicon & Metagenomics Read Simulator

Bygul is a Python 3 tool designed for simulating sequencing reads in wastewater surveillance and other metagenomic applications. It allows users to simulate complex multi-sample datasets with customizable proportions using industry-standard backends like wgsim and mason.

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🏗 Installation

Bygul requires Python 3. Since it relies on external simulators (wgsim and mason), we recommend using Conda to manage dependencies.For more info on wgsim and mason simulator please check their documentations.

Option 1: Via Conda (Recommended)

conda create -n bygul bioconda::bygul

Option 2: Via PyPI

pip install bygul

Note: Some binary dependencies (wgsim/mason) may need to be installed manually or built from source if using this method.

Option 3: Local Build from Source

git clone [https://github.com/andersen-lab/Bygul](https://github.com/andersen-lab/Bygul)
cd Bygul
pip install -e .

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🧬 Usage: Amplicon Sequencing Mode

Use this mode when simulating specific genomic regions defined by a primer set.

Basic Command

bygul simulate-proportions [SAMPLE1.fasta,SAMPLE2.fasta] --primers [primer.bed] --reference [reference.fasta] --proportions [0.8,0.2] --outdir [output_dir]

Advanced Examples

• Random Proportions & Mismatches: Simulate with random proportions and allow up to 2 SNPs in primer regions.

  bygul simulate-proportions sample1.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --outdir results/ --maxmismatch 2
  

• Switching Simulators: Use mason instead of the default wgsim.

  bygul simulate-proportions sample1.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --simulator mason
  

• Custom Error Rates & Lengths: Pass simulator-specific parameters (e.g. indel fraction -R) directly.

  bygul simulate-proportions sample1.fasta,sample2.fasta --primers primer.bed --reference reference.fasta -R 0.01
  

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🌍 Usage: Metagenomics Mode

Simulate reads from entire samples without requiring a primer BED file or a reference sequence.

Basic Metagenomics Simulation

bygul simulate-proportions sample1.fasta,sample2.fasta --outdir results/ --simulation_mode metagenomics

Metagenomics with Specific Parameters

bygul simulate-proportions sample1.fasta,sample2.fasta --proportions 0.5,0.5 --outdir results/ --simulation_mode metagenomics --simulator mason --illumina-read-length 200

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📝 Technical Notes

Parameter Handling

Bygul acts as a wrapper. While most flags are passed directly to the underlying simulators, the following are managed directly by Bygul for more realistic simulations(amplicon simulation mode only):

• --readcnt: Number of reads per amplicon.
• --wgsim_insert_size: Insert size for wgsim.
• --wgsim_read_length / --wgsim_error_rate.

To see all available backend flags, run:

wgsim --help
mason_simulator --help

Please note that some dependencies are not available through pypi. You need to install them using conda or build from source.

Amplicon sequencing mode

Example commands

Run the tool using the following command.

If you are just checking your primers without wanting to run the simulation, you can provide a multi-fasta file including all the sequences along with the primer file and the reference used to generate the primer file. This will create amplicon_stats.csv file as described below.

bygul check-primers sequences.fasta primer.bed reference.fasta

Example simualtion command

bygul simulate-proportions [SAMPLE1.fasta,SAMPLE2.fasta,..] --primers [primer.bed] --reference [reference.fasta] --proportions [0.8,0.2,..] --outdir [output_directory]

Simulate reads from different samples without defining proportions (will be assigned randomly, proportions can be found in results/sample_proportions.txt) and allowing upto 2 SNPs mistmatches in the primer regions.

bygul simulate-proportions sample.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --outdir results/ --maxmismatch 2

Simulate reads with user-defined proportions and specifing read simulator. bygul uses wgsim as a simulator but you can change it to mason.

bygul simulate-proportions sample.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --proportions 0.2,0.8 --simulator mason

Simulate reads with user-defined proportions and number of reads per amplicon.

bygul simulate-proportions sample.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --proportions 0.2,0.8 --readcnt 1000

Simulate reads with additional parameters such as base error rate, read length and indels fraction

bygul simulate-proportions sample.fasta,sample2.fasta --primers primer.bed --reference reference.fasta --proportions 0.2,0.8 --readcnt 1000 -e 0.001 -1 400 -2 400 -R 0.01

Notes

Number of reads per amplicon

It is recommended to define the number of reads per amplicon to be greater than the number of contigs in your amplicon file. This is particularly important when your primers are designed for whole genome sequencing, where each amplicon may contain a substantial number of contigs. Setting too few reads per amplicon may result in empty read files for certain amplicons, leading to incomplete simulated reads.

Primer bed file

🧬 Input BED File Format

The pipeline expects a tab-delimited BED file where the first six columns represent standard genomic coordinates (chrom, chromStart, chromEnd, name, poolName, strand). Crucially, the fourth column (name) must follow a strict naming convention to prevent downstream parsing failures in variant-calling tools: [Scheme-Name]_[AmpliconNumber]_[Direction]_[OptionalSuffix] (e.g., SARS-CoV-2_3_LEFT or SARS-CoV-2_3_LEFT_alt). To ensure structural boundaries are parsed correctly, the prefix must not contain underscores, and any optional trailing modifiers must be restricted to standard alternative tags (_alt, _ALT1) or tracking indexes (_0, _1). Multi-level pool formatting, such as SARS-CoV-2_400_1_LEFT_1, is malformed and will fail validation.. The maximum number of mismatches allowed for each primer sequence is 1 SNP. To change this number, you may use the --maxmismatches flag.

Complete set of available parameters

To learn more about how to adjust other parameters for the simulator please read the documentation for wgsim and mason simulator. Users can pass any simulator parameter directly in their command. The only parameters set through bygul are --readcnt and --wgsim_insert_size for amplicon sequencing mode.

Simulated reads output

Simulated reads from all samples are located in provided_output_path/reads.fastq

Information about amplicon dropouts

In order to find more about amplicon dropouts, please refer to provided_output_path/sample_name/amplicon_stats.csv file. Please note that primer_seq_x and primer_seq_y define the left and right primer sequence whereas left_match and right_match shows the actual sequence found in the sample for a better comparison of mismatching bases in the primer sequence. Additionally, if there are any ambiguous bases present in the matching sequence, the ambiguous_bases value returns true.

Metagenomics mode

Users can now simulate reads from different samples in a metagenomics setting without specifying a primer bed file. Providing a reference sequence is not required for this setting.

Example commands

Simulate reads from different samples without defining proportions (will be assigned randomly, proportions can be found in results/sample_proportions.txt).

bygul simulate-proportions sample.fasta,sample2.fasta --outdir results/ --simulation_mode metagenomics

Specify proportions for each sample and add other simulator specific parameters. To access simulator parameters, please read wgsim and mason documentation.

bygul simulate-proportions sample.fasta,sample2.fasta --proportions 0.5,0.5 --outdir results/ --simulation_mode metagenomics --simulator mason --illumina-read-length 200