A tool for Amplicon read simulation for waste water sequencing or other aplications. Users can easily simulate reads from mutiple samples with different proportions using the tool.
If you use this workflow in a paper, don't forget to give credits to the authors by citing the URL of this (original) https://github.com/andersen-lab/Bygul repository.
Bygul is written in python 3 but it requires wgsim and mason simulator to simulate reads.
git clone https://github.com/andersen-lab/Bygul
cd Bygul
pip install -e .
Please note that pip does not install all the requirements, some packages need to be installed via Conda or be built from source.
conda create -n bygul bioconda::bygul
pip install bygul
Please note that some dependencies are not available through pypi. You need to install them using conda or build from source.
Run the tool using the following command.
bygul simulate-proportions [SAMPLE1.fasta,SAMPLE2.fasta,..] [primer.bed] [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 primer.bed 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 primer.bed 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 primer.bed 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 primer.bed reference.fasta --proportions 0.2,0.8 --readcnt 1000 --error_rate 0.001 --read_length 400 --indel_fraction 0.001
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.
Please remember that the primer file must contain a column containing primer sequence. The maximum number of mismatches allowed for each primer sequence is 1 SNP. To change this number, you may use the --maxmismatches flag.
To learn more about how to adjust other parameters use bygul simulate-proportions --help
Simulated reads from all samples are located in provided_output_path/reads.fastq
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_mismatch_map and right_mismatch_map 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.