DNA Barcode Generator with Primer3 and BLAST

Author: Anthony Glaude from Labo-MAB, Pre. Marie Brunet

Overview

This script generates DNA barcodes by creating primers with Primer3 and assembling them into barcodes with randomly generated inserts. It performs a series of validation checks to ensure that the barcodes meet stringent criteria, including checks for Hamming distance, internal repeats, palindromes, and BLAST verification. The barcodes are BLASTed against a viral genome database and each other to avoid undesired similarities.

Features

Primer Creation with Primer3

• Size: 18–22 nucleotides
• Melting Temperature (Tm): 57–63 °C
• GC Content: 45–55%
• BLAST Verification: Primers are verified against a viral genome database to ensure no undesired similarity.
  • E-value Threshold: 0.1 (even slight similarity is rejected for primer specificity)

Barcode Assembly

• A 200nt barcode is assembled by concatenating the forward and reverse primers with a randomly generated insert in between.

Barcode Validation Checks

• Hamming Distance: At least 140 out of 200 nucleotides must differ between any two accepted barcodes.
• K-mer Check: Using a sliding window (10-mers), no identical fragments should be present across different barcodes.
• Internal Repeats: The barcode must not contain repeated k-mer sequences within itself.
• Partial Palindromes: Sequences with semi-palindromic structures are rejected (e.g., a 6-nt fragment with a minimum loop of 3 nt).

Final BLAST Verification

• The assembled barcodes are BLASTed against each other to ensure there is no undesired similarity.

Database Requirements

This script relies on a BLAST database of viral genome sequences. By default, the database is expected to be in the genome_db/viral_sequences/ directory. If you wish to use a custom genome database, follow the steps below to create and set up the database:

Creating the BLAST Database

1. Download your genome sequences: You will need a FASTA file containing the genome sequences (e.g., my_viruses.fasta). These sequences can be viral or any other type depending on your needs. You can obtain genome sequences from various sources, such as NCBI GenBank.

2. Create the BLAST database: To create the BLAST database from your FASTA file, use the makeblastdb command. This will index your genome sequences for use in BLAST searches.

   Run the following command in your terminal:

   makeblastdb -in my_viruses.fasta -dbtype nucl -out genome_db/viral_sequences

Configuration Parameters

Below are the main configuration settings you can modify in the script to customize the primer generation and barcode assembly process:

General Configuration

# === Configurations ===
N_TOTAL = 200  # Length of the final barcode (can be modified for different lengths)
INSERT_LENGTH = 160  # Length of the random insert between primers
N_PAIRS = 2  # Number of primer pairs to generate
GC_TARGET = 50  # Target GC content percentage for primer sequences
HAMMING_MIN = 140  # Minimum Hamming distance between barcodes
KMER_SIZE = 10  # Size of k-mers to check for duplicates in barcodes
output_dir = "work_repertory"  # Directory for saving results (change to desired output location)
BLAST_DB = os.path.join(output_dir, "genome_db", "viral_sequences")  # Path to the BLAST database
E_VALUE_CUTOFF = 0.1  # E-value threshold for BLAST search (controls similarity rejection)

# === Primer Generation Parameters ===
PRIMER_SIZE = 20  # (Fw & Rv) Primer size in nucleotides
PRIMER_MIN_SIZE = 18  # Minimum primer size (can be adjusted)
PRIMER_MAX_SIZE = 22  # Maximum primer size (can be adjusted)
PRIMER_OPT_TM = 60.0  # Optimal melting temperature (Tm) for primers (in °C)
PRIMER_MIN_TM = 57.0  # Minimum allowed melting temperature (Tm) for primers
PRIMER_MAX_TM = 63.0  # Maximum allowed melting temperature (Tm) for primers
PRIMER_NUM_RETURN = 1  # Number of primer pairs to return per primer design step

Requirements

• Python 3.x
• primer3-py (for primer design)
• Biopython (for sequence manipulation and BLAST parsing)
• BLAST+ command-line tools (for BLAST searches)
• shutil and os libraries (for file management)

Installation

You can install the required Python dependencies using pip or conda install

pip install primer3-py biopython

Ensure that the BLAST+ command-line tools are installed on your system. You can download them from NCBI BLAST.

Make sure that the BLAST tools are accessible from your system's PATH.

Usage

1. Run the Script: Execute the script to generate primers, assemble barcodes, and perform the necessary checks. The following files will be generated:

   • final_barcodes.fasta: Contains the generated barcodes.
   • primers.tsv: Contains primer details (forward and reverse sequences, Tm, GC%, etc.).

2. Temporary Files: The script will generate temporary BLAST files (e.g., temp_query.fasta, temp_result.xml) but will clean them up automatically after processing.

3. Customization: Adjust the following parameters to fit your experimental needs:

   • Primer size, melting temperature, GC content, and BLAST E-value threshold.
   • Barcode length (currently set to 200 nt).
   • Internal repeat, palindrome, and Hamming distance check criteria.

4. Clean-up: The script automatically deletes unnecessary temporary files to maintain a clean working directory. (Only result.xml is kept)

Example

python barcode_generator.py

This will run the script and generate the final_barcodes.fasta and primers.tsv files in the working directory.