This is for development purposes only. Please do not use this for research, public health, or diagnostic purposes because we are still developing and testing this software and it may have errors or change at any time.

StxTyper

StxTyper is used to determine stx type from nucleotide sequence. Stx (shiga-toxin) genes are found in some strains of Escherichia coli and code for powerful toxins that can cause severe illness. StxTyper is software to classify these genes from assembled sequence using a standard algorithm.

Installation

Prerequisites

C compiler and make

These generally come standard for unix systems, if not the user will need to intall make and GCC. MacOS users will need to go to the App store and install Xcode.

NCBI BLAST

StxTyper needs NCBI BLAST binaries in your path (specifically tblastn). If you don't already have BLAST installed see https://www.ncbi.nlm.nih.gov/books/NBK569861/ for instructions to install BLAST binaries.

Blast can also be installed using bioconda by first installing bioconda, then making sure to activate the environment in which it's installed.

• See the official install miniconda instructions for Linux
• See the official install miniconda instructions for MacOS

Then run:

source ~/miniconda3/bin/activate
conda create -y -c conda-forge -c bioconda -n blast blast
conda activate blast

If you install BLAST via conda in this way you will need to run conda activate blast before you can run StxTyper.

Compiling

git clone https://github.com/evolarjun/stxtyper.git
cd stxtyper
make
make test

Usage

stxtyper -n <assembled_nucleotide.fa> [<options>]

Example

stxtyper -n nucleotide.fa

Parameters

• -nucleotide <nucleotide_fasta> or -n <nucleotide_fasta> Assembled nucleotide sequence to search in FASTA format.

• --name <assembly_identifier> Add an identifier as the first column in each row of the report. This is useful when combining results for many assemblies.

• --output <output_file> or -o <output_file> Write the output to <output_file> instead of STDOUT

• --blast_bin <path> Directory to search for tblastn binary. Overrides environment variable $BLAST_BIN and the default PATH.

• -q or --quiet Suppress the status messages normally written to STDERR.

• --log <log_file> Error log file, appended and opened when you first run the application. This is used for debugging

Output

The output of StxTyper is a tab-delimited file with the following fields, all percent identity and coverage metrics are measured in proportion of amino-acids.

1. target_contig: The contig identifier from the input FASTA file
2. stx_type: The stx type called by the algorithm, for "operon = COMPLETE" it will be stx plus two characters (e.g., stx1a), for other values of operon stx_type will be stx1, stx2, or just stx if it can't resolve at all.
3. operon: What status the operon was found to be. It can be
   • COMPLETE for complete and fully typeable known stx types
   • PARTIAL for partial operons that are internal to contigs and not terminating at contig boundaries
   • PARTIAL_CONTIG_END for partial operons that could be split by contig boundaries due to sequencing or assembly artifacts
   • EXTENDED Where the coding sequence would extend beyond the stop codon for the reference protein
   • INTERNAL_STOP for Stx operons where one of the subunits has a nonsense mutation
   • FRAMESHIFT where StxTyper detected an indel in the coding sequence that would cause a frame shift in one or more of the subunits
   • COMPLETE_NOVEL a full-length stx operon that is not typeable using the current scheme
4. identity The combined percent identity for both A and B subunits
5. target_start The detected start of the alignments
6. target_stop The detected end of the alignments
7. target_strand What strand the target is on
8. A_reference The closest reference protein for the A subunit, empty if none aligned
9. A_identity The percent identity to the reference for the A subunit, empty if none aligned
10. A_coverage The percentage of the reference for the A subunit that is covered by the alignment, empty if none aligned
11. B_reference The closest reference protein for the B subunit, empty if none aligned
12. B_identity The percent identity to the reference for the B subunit, empty if none aligned
13. B_coverage The percentage of the reference for the B subunit that is covered by the alignment, empty if none aligned