Hardcoded variables to CLI

Primer_Design_SDM.py

@@ -2,7 +2,8 @@
 from Bio import SeqUtils
 from Bio.Seq import Seq
 from Bio.SeqRecord import SeqRecord
-from primer3 import calcTm
+from Bio.Data import CodonTable
+from primer3 import calc_tm # as calcTm - calcTm is deprecated
 
 import pandas as pd
 from itertools import combinations
@@ -12,6 +13,7 @@
 import re
 import math
 from pathlib import Path
+import argparse
 
 
 # Design primers in 96-well format for Site-directed mutagenesis. One mutation at a time for this script.
@@ -25,40 +27,43 @@
 ### INPUTS ###########################
 ######################################
 
-CODON_TABLE_FILE = 'Primer_Design/Codon_Table_Standard.csv'
+#CODON_TABLE_FILE = 'Primer_Design/Codon_Table_Standard.csv'
 #ORF_FILE = 'Phytase_II.txt' # This file has intentional mistakes to test the script
-ORF_FILE = 'Primer_Design/HMT.txt'   ## Change input ORF filename
-MUTATION_LIST_FILE = 'Primer_Design/HMT_Plate2.csv' ## Change input mutation csv filename. column name should be "Mutations"
+#ORF_FILE = 'Primer_Design/HMT.txt'   ## Change input ORF filename
+#MUTATION_LIST_FILE = 'Primer_Design/HMT_Plate2.csv' ## Change input mutation csv filename. column name should be "Mutations"
 
 
 ### OUTPUTS ###########################
 ######################################
 
-BASE_DIR = Path.cwd()
-print(BASE_DIR)
-OUTPUT_DIR = 'Primer_Design/Primers_HMT_Plate2' # Change output folder
-PRIMER_OUTPUT_FILE = 'HMT_Designed_primers.csv' # Change output file 1
-Forward_Primers_FILE = 'HMT_Forward_Primers_Plate2.csv' # Change output file 2
-Reverse_Primers_FILE = 'HMT_Reverse_Primers_Plate2.csv' # Change output file 3
+#BASE_DIR = Path.cwd()
+#print(BASE_DIR)
+#OUTPUT_DIR = 'Primer_Design/Primers_HMT_Plate2' # Change output folder
+#PRIMER_OUTPUT_FILE = 'HMT_Designed_primers.csv' # Change output file 1
+#Forward_Primers_FILE = 'HMT_Forward_Primers_Plate2.csv' # Change output file 2
+#Reverse_Primers_FILE = 'HMT_Reverse_Primers_Plate2.csv' # Change output file 3
 
 # Create the full path to the file ###########################
 ######################################
-Output_Path = BASE_DIR / OUTPUT_DIR / PRIMER_OUTPUT_FILE
-Output_Path.parent.mkdir(parents=True, exist_ok=True)
-Output_Path_Fwd = BASE_DIR / OUTPUT_DIR / Forward_Primers_FILE
-Output_Path_Rev = BASE_DIR / OUTPUT_DIR / Reverse_Primers_FILE
+def process_outputs(output_dir,primer_output_file,fwd_primers_file,rev_primers_file):
+    base_dir=Path.cwd()
+    Output_Path = base_dir / output_dir / primer_output_file
+    Output_Path.parent.mkdir(parents=True, exist_ok=True)
+    Output_Path_Fwd = base_dir / output_dir / fwd_primers_file
+    Output_Path_Rev = base_dir / output_dir / rev_primers_file
+    return Output_Path, Output_Path_Fwd, Output_Path_Rev
 
 ######################################
 ######################################
 
-def remind_user_to_check_constants():
+def remind_user_to_check_constants(mut_file,out_path,orf_file,codon_file_id):
     """Reminds the user to review constants and make changes if needed."""
-    print("\n⚠️  Reminder: Please check the following constants: \n")
-    print(f"  - MUTATION_FILE: {MUTATION_LIST_FILE}\n")
-    print(f"  - PRIMER_OUTPUT: {Output_Path}\n")
-    print(f"  - ORF_FILE: {ORF_FILE}\n")
-    print(f"  - CODON_TABLE: {CODON_TABLE_FILE}\n")
-    print("Modify these and more relevant values at the top of the script if necessary.\n")
+    print("\n⚠️ Reminder: Please check the following inputs for correctness: \n")
+    print(f"  - MUTATION_FILE: {mut_file}\n")
+    print(f"  - PRIMER_OUTPUT: {out_path}\n")
+    print(f"  - ORF_FILE: {orf_file}\n")
+    print(f"  - CODON_TABLE: {CodonTable.unambiguous_dna_by_id[int(codon_file_id)]}\n")
+    print("Modify these and other relevant values in CLI arguments if necessary. Run with -h for help.\n")
 
 
 def find_repeated_kmers(seq, k=16):
@@ -71,12 +76,12 @@ def find_repeated_kmers(seq, k=16):
     if repeats:
         print(f'\nWarning: {repeats} repeats of {k}bp or more in the ORF. This will affect PCR & SDM.')
 
-def read_orf_and_mutation_list():
+def read_orf_and_mutation_list(orf_file,mutation_list_file,codon_table_file_id):
     """Read the ORF sequence, mutation list, and codon table."""
-    orf_seq = Seq(Path(ORF_FILE).read_text().strip())
-    mutation_list = pd.read_csv(MUTATION_LIST_FILE)
-    codon_table = pd.read_csv(CODON_TABLE_FILE)
-    return orf_seq, mutation_list, codon_table
+    orf_seq = Seq(Path(orf_file).read_text().strip())
+    mutation_list = pd.read_csv(mutation_list_file)
+    #codon_table = CodonTable.unambiguous_dna_by_id[codon_table_file_id].tolist() #pd.read_csv(codon_table_file)
+    return orf_seq, mutation_list #, codon_table
 
 
 def validate_position(amino_acid_pos, protein_len):
@@ -97,23 +102,26 @@ def validate_mutations(mutation_list, orf_seq):
             sys.exit(f'ERROR: at null value in csv file. Format the csv file.')
 
 
-def design_primers(orf_seq, mutations, codon_table):
+def design_primers(orf_seq, mutations, codon_table_id):
     """Main primer design function."""
     primers = []
 
     for mutation in mutations['Mutations']:
         pos = int(re.search(r'\d+', mutation).group()) - 1
         validate_position(pos + 1, len(orf_seq) // 3)
 
+        # Get all possible translation table combinations for the aa resiudes in question
         original_aa, target_aa = mutation[0], mutation[-1]
-        codons = codon_table[codon_table['SingleLetter'] == target_aa]['Codon'].tolist()
+        codon_table = CodonTable.unambiguous_dna_by_id[int(codon_table_id)].forward_table
+        codons = [key for key, value in codon_table.items() if value == target_aa]
+        #codons = codon_table[codon_table['SingleLetter'] == target_aa]['Codon'].tolist()
         new_codon = find_optimal_codon(orf_seq, pos, codons)
 
         mutated_seq = orf_seq[:pos * 3] + new_codon + orf_seq[(pos + 1) * 3:]
         primer = extract_primer(mutated_seq, pos * 3)
-        tm = int(math.ceil(calcTm(str(primer), dv_conc=2, tm_method='santalucia', salt_corrections_method='owczarzy')))
+        tm = int(math.ceil(calc_tm(str(primer), dv_conc=2, tm_method='santalucia', salt_corrections_method='owczarzy')))
 
-        primers.append((mutation, primer, tm, int(SeqUtils.GC(primer)), len(primer)))
+        primers.append((mutation, primer, tm, int(SeqUtils.gc_fraction(primer)*100.0), len(primer))) #SeqUtils.GC is deprecated after Biopython 1.82 
 
     return pd.DataFrame(primers, columns=['Name', 'Sequence', 'Tm', 'GC', 'Length'])
 
@@ -146,7 +154,7 @@ def create_primer_order_file(primers):
     return pd.DataFrame(primer_order, columns=['Name', 'Sequence', 'Tm', 'GC', 'Length'])
 
 
-def separate_primers_by_type(primers):
+def separate_primers_by_type(primers, out_path_fwd, out_path_rev):
     """Separate forward and reverse primers into different CSV files."""
 
     # Create forward and reverse primer DataFrames safely
@@ -161,8 +169,8 @@ def separate_primers_by_type(primers):
     rev_primers.loc[:, 'Well'] = wells[:len(rev_primers)]
 
     # Save to CSV files
-    fwd_primers.to_csv(Output_Path_Fwd, index=False)
-    rev_primers.to_csv(Output_Path_Rev, index=False)
+    fwd_primers.to_csv(out_path_fwd, index=False)
+    rev_primers.to_csv(out_path_rev, index=False)
 
     print("\nPrimers separated for IDT order in 96-well plate.")
 
@@ -176,20 +184,44 @@ def Validate_primer_length(primer_length):
 
 if __name__ == '__main__':
     start_time = time.time()
-    remind_user_to_check_constants()
+
+    parser = argparse.ArgumentParser()
+    # MUTATION_LIST_FILE = 'Primer_Design/HMT_Plate2.csv'
+    parser.add_argument('-m', '--Mutation_List', default='Primer_Design/HMT_Plate2.csv', help="List of Mutation Names mapped to well positions")
+    # 'Primer_Design/Primers_HMT_Plate2'
+    parser.add_argument('-o', '--Output_Directory', default='Primer_Design/Primers_HMT_Plate2', help="Output directory")
+    # 'HMT_Designed_primers.csv'
+    parser.add_argument('-f', '--Primer_Output_File', default='HMT_Designed_primers.csv', help="Output file for primers assoc. characteristic data")
+    # 'HMT_Forward_Primers_Plate2.csv'
+    parser.add_argument('-fwd', '--Forward_Primers_File', default='HMT_Forward_Primers_Plate2.csv', help="Output file for Forward primers")
+    # Reverse_Primers_FILE = 'HMT_Reverse_Primers_Plate2.csv
+    parser.add_argument('-rev', '--Reverse_Primers_File', default='HMT_Reverse_Primers_Plate2.csv', help="Output file for Reverse primers")
+
+    # CODON_TABLE_FILE = 'Primer_Design/Codon_Table_Standard.csv'
+    #parser.add_argument('-c', '--Codon_Table_File', default='Primer_Design/Codon_Table_Standard.csv', help="Codon Translation Table Mapping File")
+
+    parser.add_argument('-c','--NCBI_Codon_Table_Value', default=1,help="NCBI Codon Translation Table ID Value") 
+    # ORF_FILE = 'Primer_Design/HMT.txt' 
+    parser.add_argument('-orf', '--ORF_File', default='Primer_Design/HMT.txt', help="File containing Open Reading Frame Sequence") 
+
+    args = parser.parse_args()
+
+    out_path, path_fwd, path_rev = process_outputs(args.Output_Directory,args.Primer_Output_File,args.Forward_Primers_File,args.Reverse_Primers_File)
+
+    remind_user_to_check_constants(args.Mutation_List,args.Output_Directory,args.ORF_File,args.NCBI_Codon_Table_Value)#args.Codon_Table_File)
     print(f'Working Directory: {os.getcwd()} \nProcessing...')
 
-    orf_seq, mutations, codon_table = read_orf_and_mutation_list()
+    orf_seq, mutations = read_orf_and_mutation_list(args.ORF_File,args.Mutation_List,args.NCBI_Codon_Table_Value)
 
     find_repeated_kmers(orf_seq)
     #check if provided mutations align with translation
     validate_mutations(mutations['Mutations'].tolist(), orf_seq) 
 
-    primers = design_primers(orf_seq, mutations, codon_table)
+    primers = design_primers(orf_seq, mutations, args.NCBI_Codon_Table_Value)
     Validate_primer_length(primers["Length"].tolist())
 
     primer_order = create_primer_order_file(primers)
-    primer_order.to_csv(Output_Path, index=False)
+    primer_order.to_csv(out_path, index=False)
 
-    separate_primers_by_type(primer_order)
+    separate_primers_by_type(primer_order,path_fwd,path_rev)
     print(f"\nFinished in {time.time() - start_time:.6f} seconds.")

README.md

@@ -24,6 +24,13 @@ This repository contains a set of Python scripts designed for efficient primer d
 - Python 3.x
 - Libraries: `pandas`, `itertools`, `collections`, `pathlib`, `os`, `sys`, `time`
 
+### Setup and Package Versions
+We recommend using a python virtual environment or conda environment to manage python package versions
+
+To setup with the appropriate versions, run the command:
+
+#### pip install requirements.txt
+
 ### Reference
 <details>
 <summary>If you use this tool, please cite us:</summary>

requirements.txt

@@ -0,0 +1,8 @@
+biopython==1.83
+numpy==1.24.4
+pandas==2.0.3
+primer3-py==2.2.0
+python-dateutil==2.9.0.post0
+pytz==2025.2
+six==1.17.0
+tzdata==2025.2