– Find the best linkers to use for the given primers


generate_linkers is designed to suggest primer linkers based upon the least frequently occuring base pairs (default 2) immediately upstream from the 5’ region of the primer.

A single _hits.txt file, multiple hits files, or a directory of hits.txt files can be passed as input (see the file for information about the hits.txt file format). The fasta files used to generate the _hits.txt files are also required input for this module.

The output file will contain the percentage occurance of each base at all positions in the linker and a suggested linker based on complementarity to the least frequently occurring bases.

Usage: [options]

Input Arguments:



-i, --hits_fps
Target primer hits files to generate linkers with. Separate multiple files with a colon.
-f, --fasta_fps
Fasta filepath(s). Must include all fasta sequences used to generate the hits files. Separate multiple files with a colon.


-l, --linker_len
Size of linker in base pairs. [default: 2]
-r, --all_files
Test all _hits.txt files in directory specified with -i. [default: False]
-o, --output_dir
Specify output directory for linkers summary. [default: .]
-s, --score_type
Value to use from primer hits file to determine a given primer’s amplification success. Valid choices are weighted_score, overall_mismatches, tp_mismatches. Gibbs energy scores not currently implemented [default: weighted_score]
-t, --score_threshold
If primer has score at or below this parameter, the primer amplification is considered to be successful. [default: 1.0]


A text file will be generated for each hits file containing details about the base frequency for each position of the linker and the suggested linker sequence.

Standard Example usage: [options] {-i hits_filepath [required] -f input_fasta_filepath [required]}

Change linker size to 3, manually pass 2 hits files to test, use overall mismatches for scoring: -i primer1f_hits.txt:primer2r_hits.txt -f seqs.fasta -l 3 -s overall_mismatches

Test all hits file in a target directory, put output in linkers directory: -i hits_dir/ -f seqs.fasta -o linkers/

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