SNP Mutator is a single script called snpmutator. After installation, it will be on your path, ready to use.

Quick Start

Step 1 - Create a work directory:

$ mkdir workdir
$ cd workdir

Step 2 - Gather a reference fasta file:

# For this example we will use a fasta file from our sister project, snp-pipeline
$ wget

Step 3 - Generate the mutated sequences:

# The mutated sequence files are generated in the current working directory
# -r 1, set random seed
# -n 1000, generate 1000 mutated sequences
# -s 900, nine hundred substitutions in each mutated sequence
# -i 50, fifty insertion in each mutated sequence
# -d 50, fifty deletions in each mutated sequence
# -o summary.tsv, generate a mutation summary file called summary.tsv
# -v variants.vcf, generate a VCF file of mutations
# -p 100000, choose mutations from a pool of 100000 positions
# -g 100, partition the 1000 replicates into 10 groups of 100 replicates, with each group having a separate pool of positions
# -m, create monomorphic alleles within each pool
# -M metrics, generate a metrics output file
# -R seq.fasta, generate the contatenated reference fasta file
# -F fasta, directory where the fasta replicates will be generated
$ snpmutator -r 1 -n 1000 -s 900 -i 50 -d 50 -o summary.tsv -v variants.vcf -p 100000 -g 100 -m -M metrics -R seq.fasta -F fasta NC_011149.fasta

Step 4 - Examine the results:

$ ls NC_011149_mutated_*.fasta
$ less summary.tsv
$ cat metrics

Input Files

The only input file is the reference fasta file.

Note: Multi-fasta files may produce unexpected results. All the sequences are concatenated into a single sequence. All description lines after the first are discarded.

Output Files


An optional summary file in tab-delimited format lists the positions of the mutations for each of the replicates with the original base and the resulting mutation at each position.


An optional VCF file lists the mutations in Variant Call Format.

Replicate Files

Multiple mutated replicate files are generated in a directory of your choice. Files are named with the basename of the original reference file, suffixed with _mutated_#.fasta.

For example, if the reference file name is NC_011149.fasta, the first two replicate files are named NC_011149_mutated_1.fasta and NC_011149_mutated_2.fasta.

The defline (description) of the generated fasta files is copied from the original reference fasta file, but with a suffix describing the mutations. For example, the defline suffix (mutated s=2 i=1 d=0) indicates there are two substitutions, one insertion, and zero deletions.

Reference File

An optional concatenated reference file can be generated. This is the original fasta file with all the sequences concatenated into a single sequence. All the replicates will be mutations of this file.


With the --metrics option, a file of metrics is created describing the mutated positions.


The --pool option increases the likelihood of multiple replicates having mutations at the same positions by limiting the number of positions along the genome where mutations will be introduced. The positions in the pool are choosen randomly and uniformly from the positions in the genome.


The --group option partitions the replicates into groups with each group having a different pool of eligible positions. This has the effect of creating more closely related replicates within groups and more distant replicates between groups.

Monomorphic Alleles

The --mono option ensures that when multiple replicates have a mutation at the same position, the mutation will be identical in each replicate. However, when used with the --group option, the monomorphic mutations are only within the group. Different groups of replicates may have polymorphic alleles with respect to other groups of replicates.

Command Reference

usage: snpmutator [-h] [-o FILE] [-n INT] [-s INT] [-i INT] [-d INT] [-r INT]
                  [-p INT] [-g INT] [-m] [-I SEQID] [-v FILE] [-M FILE]

Generate mutated sequence files from a reference genome. Takes a fasta file
and creates a specified number of randomly generated base substitutions,
insertions, and deletions. Outputs the mutated genomes, and optionally, a
summary file listing the mutations by position.

positional arguments:
  input_fasta_file      Input fasta file.

optional arguments:
  -h, --help            show this help message and exit
  -n INT, --num-simulations INT
                        Number of mutated sequences to generate. (default:
  -s INT, --num-substitutions INT
                        Number of substitutions. (default: 500)
  -i INT, --num-insertions INT
                        Number of insertions. (default: 20)
  -d INT, --num-deletions INT
                        Number of deletions. (default: 20)
  -r INT, --random-seed INT
                        Random number seed; if not set, the results are not
                        reproducible. (default: None)
  -p INT, --pool INT    Choose variants from a pool of eligible positions of
                        the specified size (default: 0)
  -g INT, --group INT   Group size. When greater than zero, this parameter
                        chooses a new pool of positions for each group of
                        replicates. (default: None)
  -m, --mono            Create monomorphic alleles (default: False)
  -I SEQID, --seqid SEQID
                        Output fasta description line sequence ID. Each
                        mutated output file has only one sequence. If not
                        specified, the defline id will be the id of the first
                        sequence in the input fasta file. The defline is
                        always suffixed with an annotation in this format:
                        (mutated s=900 i=50 d=50). The seq id is also written
                        to the CHROM column of the output VCF file. (default:
  -R FILE, --ref FILE   Output concatenanted reference file with no mutations,
                        but all sequences concatenanted together. All the
                        replicates will be mutations of this file. (default:
  -o FILE, --summary FILE
                        Output positional summary file. (default: None)
  -v FILE, --vcf FILE   Output VCF file. (default: None)
  -M FILE, --metrics FILE
                        Output metrics file. (default: None)
  --version             show program's version number and exit