APECtyper

In silico command line tool for typing Avian Pathogenic Escherichia coli (APEC)

Description

APECtyper is a Bash shell script that classifies Avian Pathogenic Escherichia coli (APEC) based on the revised APEC pathotyping scheme developed by Johnson et al. in prep. Preassembled partial or complete genome assemblies are run through two E. coli typing tools (namely, ECTyper and mlst) and a report is generated summarizing the results of both programs along with the APEC pathotype classification. APECtyper also screens input assemblies for 46 APEC virulence genes compiled in a custom APEC database and generates a summary report.

Dependencies

• ECTyper v1.0.0
• mlst v2.19.0
• NCBI BLAST+ blastn
  • This should already be installed as an mlst dependency.
• R

Installation

Install the latest version directly from GitHub:

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

Check Installation

Check APECtyper version:

./APECtyper.sh -v

Check that dependencies are installed in your path:

./APECtyper.sh -d

Usage

Input Requirements

• assembly in FASTA format or directory containing multiple assemblies in FASTA format (can be multiple contigs)
• directory name to output the results (to be created during run)

Command Line Options

% ./APECtyper.sh
Usage: APECtyper.sh [OPTIONS] -f [FASTA or DIR] -o [DIR]
	-h	print this usage message
	-v	print the version
	-r	print citation
	-d	check for dependencies
	-f	FASTA contig file or directory containing multiple FASTA files
	-o	output directory
        -i      minimum blast % identity [default: 90]
	-c      minimum blast % coverage [default: 90]
	-t      number of threads to use [default: 1]
	-s      combine reports from multiple samples into single TSV file

Basic Usage

Input is a single FASTA file:

./APECtyper.sh -f data/assemblies/sample1.fasta -o sample1_output

Input is a directory containing multiple FASTA files:

./APECtyper.sh -f data/assemblies -o multisample_output

Output

Within the user-defined output directory, there will be three directories – serotype, mlst, and blast – containing the individual output files from ECTyper, mlst, and blastn, respectively. There will also be two TSV files per sample:

• pathotype_results_[SAMPLE].tsv: A tab-separated file summarizing the outputs from ECtyper and mlst, as well as the pathotype classification.
• blast_results_[SAMPLE].tsv: A tab-separated summary of the blastn results against the custom APEC virulence gene database.
• report.log: A log file for summary report generation.

If the -s flag is included when running APECtyper.sh, two additional files will be generated that are compilations of all blast reports and all pathotype reports: blast_results_summary.tsv and pathotype_results_summary.tsv.

Pathotype output

The pathotype_results_[SAMPLE].tsv file has the following columns:

Column	Example	Description
Sample	sample1	Name of input assembly file with file extension removed
Species	Escherichia coli	Species identified by ECTyper
Serotype	O25:H4	Serotype identified by ECTyper
SerotypeQC	PASS (REPORTABLE)	QC message produced by ECTyper (See ECTyper page for a description of all possible QC codes)
ST	131	Sequence type identified by mlst
APEC_plasmid	Present	Whether or not APEC plasmid markers hlyF and ompT were found
Pathotype	High Risk APEC	Pathotype classification (possible values include High Risk APEC, APEC, High Risk non-APEC, non-APEC, or Not E. coli) *

*NOTE: Occasionally, the ST or O-type cannot be determined. In these situations, there will be an added note in the 'Pathotype' column indicating that it is unknown whether the isolate is 'High Risk'.

APEC virulence gene output

The blast_results_[SAMPLE].tsv file has the following columns:

Column	Example	Description
Sequence	contig00218	Name of input assembly or specific assembly contig
Gene	papC|Pap_pili|CP000468.1	APEC virulence gene name
GeneLength	2520	Length of gene (in bp)
AlignmentLength	2520	Length of sequence overlap (in bp)
Mismatches	1	Number of mismatches in the alignment
Gaps	0	Number of gaps in the alignment
SequenceStart	2955	Start of alignment in sequence
SequenceEnd	5474	End of alignment in sequence
GeneStart	1	Start of alignment in gene
GeneEnd	2520	End of alignment in gene
Identity	99.96	Percent of identical matches
Evalue	0	Expect value (see definition here)
Bitscore	4649	Bit score
Coverage	100	Percent coverage of the gene ( (AlignmentLength - Gaps) / GeneLength * 100 )

Citations

./APECtyper.sh -r

If you use APECtyper in your work, please cite:

• Johnson TJ, Miller EA, Flores-Figueroa C, Munoz-Aguayo J, Cardona C, Fransen K, Lighty M, Gonder E, Nezworski J, Haag A, Behl M, Kromm M, Wileman B, Studniski M, Strain E, McDermott P, Singer RS. Refining the definition of the avian pathogenic Escherichia coli (APEC) pathotype through inclusion of high-risk clonal groups. Poultry Science (2022) 101(10):102009. doi: 10.1016/j.psj.2022.102009

Please also cite:

• Bessonov K, Laing C, Robertson J, Yong I, Ziebell K, Gannon VPJ, Nichani A, Arya G, Nash JHE, Christianson S. ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data. Microb Genom. (2021) 7(12):000728. doi: 10.1099/mgen.0.000728
• Seemann T, mlst Github https://github.com/tseemann/mlst
• Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. (2018) 3:124. doi: 10.12688/wellcomeopenres.14826.1