Probe generation

Generation of nucleotide probes

On this page we describe the tool usage for oligonucleotide generation

Warning: This tool is under active development.

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Table of Contents

1. Algorithm Overview
2. Algorithm Steps
3. Preparation Step
4. Main Usage
5. Contributing
6. License
7. Contact

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Algorithm Overview

PROBEst employs a wrapped evolutionary algorithm to generate and optimize nucleotide probes. The workflow integrates Primer3, BLASTn, and a mutation module to iteratively refine probes based on user-defined criteria for universality and specificity. The AI-driven corrections further enhance the probe quality by evaluating and optimizing the probe set.

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Algorithm Steps

1. Select File for Probe Generation

   • Choose the primary file that will be used to generate the probe.

2. Select Files for Universality Check

   • Identify and select files that will be used to assess the universality of the probe.

3. Select Files for Specificity Check

   • Identify and select files that will be used to evaluate the specificity of the probe.

4. Select Layouts

   • Determine the layouts that will be utilized during the probe generation process.

5. Run Wrapped Evolutionary Algorithm

   • Execute the following steps within the evolutionary algorithm:

   a. Primer3 Generation

   • Generate primers using the Primer3 tool.

   b. BLASTn Check

   • Perform a BLASTn check to ensure the generated probes are suitable.

   c. Parsing

   • Parse the results from the BLASTn check to extract relevant information.

   d. Mutation in Probe

   • Introduce mutations in the probe based on the parsed data to optimize performance.

   e. AI Corrections

   • Probe evaluation based on AI optimizing function.

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Preparation Step

pipeline.py relies on pre-prepared BLASTn databases. To create the required true_base, false_base, and contig_table, you can use the following script:

bash scripts/generator/prep_db.sh \
  -n {database_name} \
  -c {contig_name} \
  -t {tmp_dir} \
  [fasta_files]

Arguments:

• -n {database_name}:
  Name of the output BLAST database (required).
• -c {contig_name}:
  Output file to store contig names and their corresponding sequence headers (required).
• -t {tmp_dir}:
  Temporary directory for intermediate files (optional, defaults to ./.tmp).
• [fasta_files]:
  List of input FASTA files (gzipped or uncompressed).

This script prepares the necessary files for running the pipeline.py tool. Ensure that the input FASTA files are correctly formatted and accessible.

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Main Usage

PROBEst can be run using the following command:

python pipeline.py [-h] \
  -i {INPUT} \
  -tb {TRUE_BASE} \
  -fb [FALSE_BASE [FALSE_BASE ...]] \
  -c {CONTIG_TABLE} \
  -o {OUTPUT}

Key Arguments:

• -i INPUT: Input FASTA file for probe generation.
• -tb TRUE_BASE: Input BLASTn database path for primer adjusting.
• -fb FALSE_BASE: Input BLASTn database path for non-specific testing.
• -c CONTIG_TABLE: .tsv table with BLAST database information.
• -o OUTPUT: Output path for results.
• -t THREADS: Number of threads to use.
• -a ALGORITHM: Algorithm for probe generation (FISH or primer).

For a full list of arguments, run:

python pipeline.py --help