In silico PCR command line tool for typing Escherichia coli ST131

ST131Typer is a simple Bash shell script that utilizes the SeqKit function seqkit amplicon to detect Escherichia coli sequence type (ST) 131 and characterize its key subclones from preassembled partial or complete genome assemblies.

This tool is the in silico version of the in vitro multiplex PCR assays developed by Johnston et al. in prep. The lab assay allows resolution of 15 distinctive molecular subsets within ST131, including 3 within ST131 clade A (i.e. the H41 subclone), 5 within clade B (i.e. the H22 subclone), and 7 within clade C (i.e. the H30 subclone), including subclones C0 (i.e. H30S: 2 subsets), C1 and C1-M27 (i.e. H30R1: 2 subsets), and C2 (i.e. H30Rx: 3 subsets).

Specifically, this tool uses primer sequences to target distinctive nucleotide polymorphisms or sequences in:

• mdh36
• gyrB47
• trpA72
• sbmA
• plsB
• nupC
• rmuC
• kefC
• ybbW
• O16 and O25b rfb variants
• five key fimH alleles (fimH22, fimH27, fimH30, fimH35, and fimH41)
• two fliC alleles (H4 and H5)
• (subclone-specific) fluoroquinolone resistance-associated parC allele
• (subclone-specific) prophage marker

If the target polymorphism or sequence is found in a WGS assembly, the resulting target sequence (the sequence that lies between and includes the primer pair), or "amplicon", is output.

ST131Typer is only as accurate as the quality of the input sequences. Less complete assemblies increase the likelihood that a target sequence is fragmented or incomplete and therefore not identified by ST131Typer. Be aware that ST131Typer will not consider a target present if:

• The target spans two assembly contigs/scaffolds (i.e. the forward and reverse primers are on different contigs/scaffolds).
• Only one primer of a pair is identified.
• The primer sequences do not match 100% with the target.

BLAST-based tools, such as those on the Center for Genomic Epidemiology (CGE) website, utilize curated databases of complete allele sequences and do not require a 100% match between the input assembly and the allele sequences. Since these tools are simply reporting the top BLAST hit(s), they are less affected by incomplete genome assemblies or allelic variation in primer regions than ST131Typer. The benefit of using ST131Typer over publicly available BLAST-based tools is that it provides typing resolution beyond just serotype and fimH type.

• SeqKit (>= v0.14.0)
  • Go to SeqKit's Installation Page for install options.

This will install the latest version directly from GitHub:

git clone https://github.com/JohnsonSingerLab/ST131Typer.git

Change permissions to make ST131Typer.sh executable:

cd ST131Typer
chmod +x ST131Typer.sh

Ensure the desired ST131Typer version is installed:

./ST131Typer.sh -v

Check that the dependency, SeqKit, is installed in your path:

./ST131Typer.sh -c

• assembly in FASTA format or directory containing multiple assemblies in FASTA format; assemblies can be in multiple contigs
• directory to output the results into

% ./ST131Typer.sh
Usage: ST131Typer.sh [OPTIONS] -i [FASTA or DIR] -o [DIR]
	-h		print this message
	-v		print the version
	-c		check SeqKit is in path
	-i		fasta contigs file or directory containing multiple files
	-o		output directory
  	-r    		prints citation

Single FASTA file:

% ./ST131Typer.sh -i data/assemblies/BS448.fasta -o example_output

Directory containing multiple FASTA files:

% ./ST131Typer.sh -i data/assemblies -o example_output

Assembly FASTA files of strains representing 14 of the 15 ST131 PCR profile types described in Johnston et al. in prep were downloaded from EnteroBase's Escherichia/Shigella Database and can be found in the data/assemblies directory.

Within the user-defined output directory, there will be two items:

• summary.txt: A simple tab-separated summary of the results
• seqkit_outputs: A directory containing the output(s) of seqkit amplicon in BED format, one file for each assembly file input.

The summary.txt file is a tab-separated results summary with one line for each assembly file input.

^* Within the target columns, there are five possible entries:

./ST131Typer.sh -r

If you use ST131Typer in your work, please cite:

Johnston, BD, Gordon, DH, Burn, S, Johnson, TJ, Weber, BP, Miller, EA, and Johnson, JR. Novel Multiplex PCR Method for High-Resolution Subclonal Assignment and Characterization of Escherichia coli Sequence Type 131 Isolates.

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