In silico taxonomic classification of Bacillus cereus group isolates using assembled genomes

BTyper3 is a command-line tool for taxonomically classifying Bacillus cereus group genomes using a standardized nomenclature.

The program, as well as the associated databases, can be downloaded from https://github.com/lmc297/BTyper3.

Post issues at https://github.com/lmc297/BTyper3/issues.

For more information, check out the BTyper3 wiki at https://github.com/lmc297/BTyper3/wiki.

For more information, check out the BTyper3 wiki

To create a conda environment named btyper3 and install BTyper3 and all of its dependencies:

1. Install conda, if necessary
2. Create a new environment named btyper3 by running the following command from your terminal:

   conda create -n btyper3

3. Activate your btyper3 environment by running the following command from your terminal:

   conda activate btyper3

4. Install BTyper3 by running the following command from your terminal:

   conda install bioconda::btyper3

5. You can now run btyper3! Run the following command from your terminal to view all btyper3 options, or check the BTyper3 wiki for details:

   btyper3 --help

6. When you're done with BTyper3, you can deactivate the btyper3 environment by running the following:

   conda deactivate

1. To run BTyper3, please download and install the following dependencies, if necessary:

   • Python 3
   • BLAST v2.9.0+ and up

2. Add BLAST+ to your path, if necessary (to check if BLAST+ is in your path, try running makeblastdb -h and tblastn -h from your command line; you should get a help message for each command, with no error messages)

3. Install via pip (this will download required Python dependencies as well):

   pip install btyper3  

The primary function of BTyper3 is to allow users to taxonomically classify B. cereus group genomes using a standardized nomenclature (see here and here for details regarding how the standardized nomenclature was constructed, and how it compares to historical typing methods, respectively). However, we understand that some users may also want to compare their B. cereus group genomes to the type strain genomes of published B. cereus group species. Thus, in BTyper3 v3.2.0, we have added the --ani_typestrains option, which calculates ANI values between a query genome and the genomes of all published B. cereus group species type strains and reports the type strain that produces the highest ANI value.

The type strain genomes used by BTyper3's --ani_typestrains option correspond to the species discussed in Figure 2 of our taxonomy review, plus species published after the review was published (i.e., Bacillus sanguinis, Bacillus paramobilis, and Bacillus hominis, added in v3.2.0; "B. arachidis" and B. rhizoplanae, added in v3.3.0; "B. pretiosus", added in v3.4.0). Within the standardized taxonomy that BTyper3 uses for genomospecies assignment:

• All members of Bacillus sanguinis (type strain RefSeq Assembly Accession GCF_018332475.1) belong to B. mosaicus (i.e., B. sanguinis is not considered a novel species in the standardized taxonomy)

• All members of Bacillus paramobilis (type strain RefSeq Assembly Accession GCF_018332495.1) belong to B. mosaicus (i.e., B. paramobilis is not considered a novel species in the standardized taxonomy)

• All members of Bacillus hominis (type strain RefSeq Assembly Accession GCF_018332515.1) belong to B. mycoides (i.e., B. hominis is not considered a novel species in the standardized taxonomy)

• "Bacillus arachidis" (type strain RefSeq Assembly Accession GCF_017498775.1) replaces the putative genomospecies previously referred to as "Unknown Species 17" in the standardized taxonomy

• Bacillus rhizoplanae (type strain RefSeq Assembly Accession GCF_917563915.1) represents a novel genomospecies within the standardized taxonomy and has been added to the database

• All members of "Bacillus pretiosus" (type strain RefSeq Assembly Accession GCF_025916425.1) belong to B. mosaicus (i.e., "B. pretiosus" is not considered a novel species in the standardized taxonomy)

Importantly, B. cereus group species are often proposed in the literature using unstandardized approaches (e.g., varying genomospecies thresholds, which may produce overlapping genomospecies). We have added the type strain comparison method in BTyper3 v3.2.0, as users may still want to compare a query genome with the type strains of published B. cereus group species. However, interpret results with caution, as some B. cereus group genomes may belong to multiple species using type strain genomes.

For more information, check out our:

• Review of B. cereus group taxonomy/nomenclature

• Standardized nomenclature for the B. cereus group

• Comparison of our standardized nomenclature to other B. cereus group typing methods (e.g., MLST, panC, ANI-based comparisons to species type strain genomes)

Carroll, Laura M., Martin Wiedmann, Jasna Kovac. 2020. "Proposal of a Taxonomic Nomenclature for the Bacillus cereus Group Which Reconciles Genomic Definitions of Bacterial Species with Clinical and Industrial Phenotypes." mBio 11(1): e00034-20; DOI: 10.1128/mBio.00034-20.

Carroll, Laura M., Rachel A. Cheng, Jasna Kovac. 2020. "No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus group with Historical Typing Methods." Frontiers in Microbiology 11: 580691; DOI: 10.3389/fmicb.2020.580691.

For detailed information, check out the BTyper3 wiki

btyper3 -i [fasta] -o [output directory] [options...]

For help, type btyper3 -h or btyper3 --help

For your current version, type btyper3 --version

btyper3 -i /path/to/genome.fasta -o /path/to/desired/output_directory

btyper3 -i /path/to/genome.fasta -o /path/to/desired/output_directory --fastani_path /path/to/FastANI_executable/fastANI

btyper3 -i /path/to/genome.fasta -o /path/to/desired/output_directory --ani_geneflow True

btyper3 -i /path/to/mlst.fasta -o /path/to/desired/output_directory --ani_species False --ani_subspecies False --ani_typestrains False --virulence False --bt False --panC False --download_mlst_latest True

btyper3 -i /path/to/panC.fasta -o /path/to/desired/output_directory --ani_species False --ani_subspecies False --ani_typestrains False --virulence False --bt False --mlst False

btyper3 -i /path/to/plasmid.fasta -o /path/to/desired/output_directory --ani_species False --ani_subspecies False --ani_typestrains False --mlst False --panC False

Disclaimer: BTyper3 is pretty neat! However, no tool is perfect, and BTyper3 cannot definitively prove whether an isolate is pathogenic or not. As always, interpret your results with caution. We are not responsible for taxonomic misclassifications, misclassifications of an isolate's pathogenic potential or industrial utility, and/or misinterpretations (biological, statistical, or otherwise) of BTyper3 results.