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/MixAmp repository.
Mixamp is written in python 3 but it requires wgsim and mason simulator to simulate reads.
git clone https://github.com/andersen-lab/MixAmp
cd mixamp
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.
pip install git+https://github.com/andersen-lab/MixAmp- Create a conda environment as mixamp and install the dependencies:
conda create -n mixamp
conda activate mixamp
conda env update --file environment.yml
Run the tool using the following command.
mixamp 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.
mixamp simulate-proportions sample.fasta,sample2.fasta primer.bed reference.fasta --outdir results/ --maxmismatch 2
Simulate reads with user-defined proportions and specifing read simulator. Mixamp uses wgsim as a simulator but you can change it to mason.
mixamp 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.
mixamp 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
mixamp 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 mixamp 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. This file will have right/left primer matching coordinates as zero if no matches found.