Table of Contents

  1. Introduction
  2. Prerequisites
  3. Inputs
  4. Running the Script
  5. Command Line Arguments
  6. Outputs
  7. Interpreting the Results
  8. Troubleshooting
  9. Additional Resources

Introduction

This script processes STEC alleles with both A and B subunits. It performs BLAST analyses to identify the best matching alleles for each subunit, detects novel alleles, and generates reports summarizing the results. This is essential for accurate subunit-level typing and characterization of Shiga toxin-producing Escherichia coli (STEC).

Prerequisites

Before running the script, ensure you have the following:

  1. Query files in FASTA format containing nucleotide or amino acid sequences.
  2. Allele database files for the relevant subunits (A and B) in FASTA format.

Inputs

The script requires the following inputs:

  1. Query files in FASTA format.
  2. Allele database files for the A and B subunits (e.g., Stx1A_aa_*.fasta, Stx1B_aa_*.fasta, Stx2A_aa_*.fasta, Stx2B_aa_*.fasta).
  3. Optionally, a directory containing split AA allele databases for subunit-level BLAST analyses.

Running the Script

To run the script, use the following command:

stec.py stec_combined_subunits --allele_path /path/to/allele_folder --report_path /path/to/output_folder --query_path /path/to/query_folder --blast_mode blastn --split_aa_db_dir /path/to/split_aa_db_dir

Command Line Arguments

The script accepts the following command line arguments:

  • -h, --help: Show this help message and exit.
  • --version: Show program's version number and exit.
  • -a, --allele_path: Specify the path to the folder containing allele files. Default: alleles in the current working directory.
  • -r, --report_path: Specify the path to the folder for output reports. Default: reports in the current working directory.
  • -q, --query_path: Specify the path to the folder containing query files in FASTA format. Default: query in the current working directory.
  • --blast_mode: Choose BLAST mode: blastn, tblastx, blastx, or blastn+tblastx. Default: blastx.
  • --preliminary: Run a preliminary screen using blastn (do not run additional downstream analyses).
  • --split_aa_db_dir: Path to directory containing split AA allele databases (required for full analysis).
  • -n, --num_alignments: Number of alignments to return. Default: 10.
  • -c, --cutoff: Percent identity cutoff. Default: 99.0.
  • -v, --verbosity: Set the logging level. Options: debug, info, warning, error, critical. Default: info.

Outputs

The script generates the following output files:

  • novel_alleles.fasta: FASTA file containing all novel alleles discovered.
  • stec_combined_nt_report.tsv: TSV report summarizing nucleotide-level matches and novel alleles.
  • stec_combined_aa_report.tsv: TSV report summarizing amino acid-level matches and novel alleles.
  • stec_combined_nt_aa_report.tsv: Combined report for both nucleotide and amino acid analyses.
  • Individual FASTA files for novel subunits and operons, as appropriate.

Interpreting the Results

The main reports (stec_combined_nt_report.tsv, stec_combined_aa_report.tsv, and stec_combined_nt_aa_report.tsv) summarize the best matches for each query, percent identity, and any novel alleles detected. Additional notes may indicate partial matches, ambiguous bases, or gap events.

Troubleshooting

If you encounter issues:

  • Ensure all input files are in the correct format and location.
  • Confirm that BLAST+ is installed and accessible from your command line.
  • Check that the split AA database directory contains the required subunit FASTA files.
  • Review error messages for missing files or permission issues.

Additional Resources

For more information, see the stec_combined_subunits documentation or open an issue on GitHub.