5. Frequently Asked Questions

• What's the difference between BTyper3 and the original BTyper?

BTyper3 is a completely novel tool for determining the identity of B. cereus group isolates using WGS data and a standardized taxonomic nomenclature. Unlike its predecessor, which focused on gene detection and required a signifcant amount of user knowledge/interpretation, BTyper3 aims to provide taxonomic classifications at a whole-genome scale which require minimal user interpretation/prior domain-specific knowledge of the B. cereus group.

• Why is it named BTyper3?

BTyper3 uses python3; python2, which the original BTyper relied on, will not be maintained in the future. Additionally (and conveniently!), the most recent version of the original BTyper was version 2, making BTyper3 a logical next step.

• Why doesn't BTyper3 perform rpoB allelic typing like the original BTyper?

When developing BTyper3, we found that the resolution rpoB provided was lower than that of MLST and panC. We thus opted to not currently include it in BTyper3 (this may change in the future, but for now, see the original BTyper if you'd like to perform rpoB allelic typing).

• Why doesn't BTyper3 perform antimicrobial resistance (AMR) determinant detection like the original BTyper?

The AMR determinant detection method implemented in the original BTyper was difficult to maintain and wasn't B. cereus group-specific; we thus considered it to fall outside of the scope of BTyper3, and we opted to not include it. In addition to the original BTyper, there are many AMR determinant detection methods available now, including some that do effectively the same thing as the original BTyper.

• Can I analyze BTyper3 output files with BMiner?

Sadly, BMiner currently does not support BTyper3 files as input, as BTyper3 and the original BTyper (version 2.X.X and earlier) produce files in different output formats. BTyper3 was developed to produce output files in a standard tabular format, which we anticipate will be easier for users to work with.

To aggregate all BTyper3 results from a single run into a single tab-separated table which can then be loaded into R, Excel, etc., simply:

1. Move to your BTyper3 output directory: cd < name of the directory you supplied to btyper3 -o >
2. Concatenate BTyper3 final results files into a single file, here named btyper3_all_results.txt (the -v option excludes the file headers): cat btyper3_final_results/*_final_results.txt | grep -v "#filename" > btyper3_all_results.txt

• Can I use sequencing reads (e.g., Illumina reads) as input for BTyper3?

No; BTyper3 requires an assemlbed genome in FASTA format. While the original BTyper had the option to use SPAdes for genome assembly, we opted to not include it in BTyper3, as it became time-consuming to maintain, without adding significant value to users.

• Can I use multiple B. cereus group genomes in a single FASTA file (e.g., an assembled metagenome) as input for BTyper3?

No; BTyper3 requires whole genomes of single B. cereus group isolates, as it relies on ANI-based methods.

• Can I use partial nucleotide sequences (e.g., plasmid sequences, MLST genes, panC alleles, etc.) as input for BTyper3?

Yes, but only if you set all ANI-based methods to False (i.e., make sure to include --ani_species False --ani_subspecies False in your command), as ANI-based methods require whole genomes.

To perform seven-gene MLST only, using the latest version of the PubMLST B. cereus database, use the following command:

btyper3 -i [fasta] -o [output directory] --ani_species False --ani_subspecies False --virulence False --bt False --panC False --download_mlst_latest True

To perform panC group assignment only, use the following command:

btyper3 -i [fasta] -o [output directory] --ani_species False --ani_subspecies False --virulence False --bt False --mlst False

To detect virulence factors and Bt toxin-encoding genes in a nucleotide sequence (e.g., a plasmid sequence), use the following command:

btyper3 -i [fasta] -o [output directory] --ani_species False --ani_subspecies False --mlst False --panC False

• Can I use WGS data from organisms that don't belong to the Bacillus cereus group?

No; BTyper3 is a taxonomic tool which relies on ANI-based methods relative to B. cereus group genomes. Results for distant species won't be interpretable.