toast-amplicon

TOAST (Tuberculosis Optimized Amplicon Sequencing Tool)

Here we introduced TOAST software tool aimed at addressing the challenges in amplicon primer design for TB sequencing leveraging the package primer3 for Tm, homopolymers, hairpins, homodimers considerations and in-house pipeline for avoiding heterodimer, alternative binding. This automated tool in takes user defined SNP priority for amplicon coverage and outputs designed amplicon primers with respective Tm and primer coordinates and sequence with capability of focusing on specific genes and taking into account of spoligotypes

Workflow

Before running the tool

*Decide on SNP priority by modifying the SNP priority file (mutation_priority_example.csv - can be found in github) *Decide on amplicon size

Dependencies

• "pandas",
• "numpy",
• "plotly",
• "rich_argparse",
• "tabulate",
• "primer3-py >= 2.0.1"

1. Estimate amplicon number needed for coverage (amplicon_no function)
   • Example:

    toast amplicon_no -a 800 -op ./cache/Amplicon_design_output -g
   

2. Run amplicon design (design function)
   • Example:

   toast design -op ./cache/Amplicon_design_output -a 400 -sn 1 -sg rpoB,katG -nn 40 
   
   toast design -op ./cache/Amplicon_design_output -a 400 -sn 1 -sg rpoB,katG -nn 25
   
   toast design -op ./cache/output -a 1000 -nn 4 -ud ./cache/test_df.csv
   
   toast design -op ./cache/output -a 1000 -nn 26
   
   toast design -op ./cache/Amplicon_design_output -a 400 -sn 1 -sg rpsL -nn 0 -ud ./cache/test_df.csv
   

3. Check amplicon design using coverage plot (plotting function)
   • Example:

   toast plotting -ap ./toast/Amplicon_design_output/Primer_design-accepted_primers-23-400.csv -rp ./toast/db/reference_design.csv -op ./cache/Amplicon_design_output -r 400
   

Primer3 Configuration Parameters (default file: db/default_primer_design_setting.txt)

• PRIMER_NUM_RETURN: Number of primer pairs to return.
• PRIMER_PICK_INTERNAL_OLIGO: Flag to pick internal oligos (0 for no, 1 for yes).
• PRIMER_INTERNAL_MAX_SELF_END: Maximum self-complementarity score for internal oligos.
• PRIMER_MIN_SIZE: Minimum primer size in bases.
• PRIMER_MAX_SIZE: Maximum primer size in bases.
• PRIMER_MIN_TM: Minimum melting temperature (Tm) for primers in °C.
• PRIMER_MAX_TM: Maximum melting temperature (Tm) for primers in °C.
• PRIMER_MIN_GC: Minimum GC content in percent for primers.
• PRIMER_MAX_GC: Maximum GC content in percent for primers.
• PRIMER_MAX_POLY_X: Maximum length of mononucleotide repeats in primers.
• PRIMER_INTERNAL_MAX_POLY_X: Maximum length of mononucleotide repeats in internal oligos.
• PRIMER_SALT_MONOVALENT: Concentration of monovalent salts (e.g., Na+, K+) in mM.
• PRIMER_DNA_CONC: Concentration of DNA template in nM.
• PRIMER_MAX_NS_ACCEPTED: Maximum number of unknown bases (N's) accepted in primers.
• PRIMER_MAX_SELF_ANY: Maximum overall self-complementarity score for primers.
• PRIMER_MAX_SELF_END: Maximum 3' end self-complementarity score for primers.
• PRIMER_PAIR_MAX_COMPL_ANY: Maximum overall complementarity score between primer pairs.
• PRIMER_PAIR_MAX_COMPL_END: Maximum 3' end complementarity score between primer pairs.
• PRIMER_PRODUCT_SIZE_RANGE: Range of acceptable primer product sizes (e.g., "100-300").

Example format of the user defined files can be found in user_defined_files/ folder:

• Configuration Parameters file: default_primer_design_setting.txt
• User input primer file: user_input_primer.csv

Ouput file format

<filetype>-<number of total amplicon designed>-<minimum amplicon size>-<maximum amplicon size>-<step size>-<number of amplicon for each size>

5 different files are produced

• Primer_design-accepted_primers: All detailed information about the designed amplicons
• Amplicon_importance: Number of SNP coverd by each amplicon
• Amplicon_mapped: bed file can be used to visualised the amplicon on genome using tools such as igv
• SNP_inclusion: shows SNP covered
• Gene_covereage: show percentage of each gene covered

Specific mutation (Mutation Priority)file format: Essentially all you need would be the genome position (genome_pos). Other columns are needed but you could used imputed values like below if unknown. The complete example Mutation priority csv can be found in Github：mutation_priority_example.csv

sample_id	genome_pos	gene	change	freq	type	sublin	drtype	drugs	weight
sample_1	321168	gene_1	change_1	1	-	-	-	-	1
sample_2	551767	gene_2	change_2	1	-	-	-	-	1
sample_3	1017188	gene_3	change_3	1	-	-	-	-	1
sample_4	1119158	gene_4	change_4	1	-	-	-	-	1
sample_5	1119347	gene_5	change_5	1	-	-	-	-	1
sample_6	1414872	gene_6	change_6	1	-	-	-	-	1

You can manually eddit this for though a script (Github：mutation_priority_gen.py) can also be found to generate a file like the above:

example usage:

python mutation_priority_gen.py --positions "322168,553767,1077188" --output <output_path.csv>