ST131Typer

In silico PCR command line tool for typing Escherichia coli ST131

Description

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.

⚠️ A word of caution...

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.

Requirements

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

Installation

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

Check Installation

Ensure the desired ST131Typer version is installed:

./ST131Typer.sh -v

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

./ST131Typer.sh -c

Usage

Input Requirements

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

Command Line Options

% ./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

Example Usage

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.

PCR Type	Representative Strain	Clade	O:H Type	fimH Allele	Description
1	MVAST020	A	O16:H5	H41	H41 / A (O16:H4 variant)
2	BS488	A	O25b:H4	H41	H41 / A (O25b:H4 variant)
3	M670745	A	O25b:H5	H41	H41 / A (O25b:H5 variant)
4	H17	B0	O25b:H4	H27	H27 / B0
5	JJ1897	B0	O25b:H4	H22	H22 / B0
6	JJ1969	B1	O25b:H4	H22	H22 / B1
7	ZH071	B1	O25b:H4	H94	H94 (non-H22) / B1
8	G199	B1	O25b:H4	H30	H30 / B1
9	CD306	C0	O25b:H4	H30	H30S / C0
10	BS448	C0	O25b:H4	H30	H30S / C0 (parC E84V and FQ-R)
11	JJ2193	C1	O25b:H4	H30	H30R1 non-C1-M27 / C1
12	U024	C1-M27	O25b:H4	H30	H30R1 C1-M27 / C1-M27
13	JJ1886	C2	O25b:H4	H30	H30Rx / C2
14	U004	C2	O25b:H4	H35	H30Rx (fimH35) / C2
15	Not Available	C2	ONT:H4	H30	H30Rx / C2 (non-O-typable)

Output

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.

Column	Description	Possible Values
Sample	Name of assembly file
PCR_Profile_Type	Numerical ST131 PCR profile type from Johnston et al. in prep	1-15, "Unknown", or "non-ST131"
Clade	ST131 clade designation	A, B0, B1, C0, C1, C1-M27, or C2
O_type	O antigen	O16 or O25b
H_type	H antigen	H4 or H5
fimH	fimH allele	fimH22, fimH27, fimH30, fimH35, or fimH41
Description	Description of the ST131 subclone/clade associated with the ST131 PCR profile type
mdh36	Length (bp) of mdh36 target identified	~275 *
gyrB47	Length (bp) of gyrB47 target identified	~138 *
trpA72	Length (bp) of trpA72 target identified	~487 *
rfb_O16	Length (bp) of rfb O16 allele target identified	~732 *
rfb_O25b	Length (bp) of rfb O25b allele target identified	~557 *
fliC_H4	Length (bp) of fliC H4 allele target identified	~199 *
fliC_H5	Length (bp) of fliC H4 allele target identified	~614 *
fimH22	Length (bp) of fimH22 allele complex target identified	~279 *
fimH27	Length (bp) of fimH27 allele complex target identified	~405 *
fimH30	Length (bp) of fimH30 allele complex target identified	~350 *
fimH35	Length (bp) of fimH35 allele complex target identified	~500 *
fimH41	Length (bp) of fimH41 allele complex target identified	~92 *
plsB	Length (bp) of plsB target identified	~628 *
nupC	Length (bp) of nupC target identified	~498 *
kefC	Length (bp) of kefC target identified	~238 *
rmuC	Length (bp) of rmuC target identified	~353 *
prophage	Length (bp) of the prophage marker target identified	~822 *
sbmA	Length (bp) of sbmA target identified	~64 *
ybbW	Length (bp) of ybbW target identified	~194 *
parC_E84V	Length (bp) of parC E84V target identified	~107 *

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

Character	Meaning
[NUM]	Length (bp) of target (sequence between and including the primer pair)
*[NUM]*	Length (bp) of target falls outside +/- 5% of the expected length
NA	Not applicable to the typing of the sample (i.e. non-ST131 strain; clade B- and C-associated primer pairs are not relevant for typing of clade A isolates)
NF	Not found; primer pair was not identified or was not identified on the same contig
NT	Non-typable; O-type, H-type, fimH-type or clade could not be determined. Could be due to missing target or the presence of multiple conflicting targets.

Citation

./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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