greenkidneybean / dms_primer_design

DMS Primer Design

Code for designing primers used to generate libraries of genetic variants.

The primer_design.py script requires a config.yaml and .gb file for a gene of interest, and outputs a .tsv file of doped primers. This script specifically designs primers that generate single-residue missense vairants that are attained by a single nucleotide change (or SNP) from the original gene of interest sequence. Example inputs are provided in the test_data directory. Variants are generated across "tiles" of the coding sequence that are listed as Features in the .gb and listed under "variant_windows" in the config.yaml. Note that the variant codons are flanked with synonymous changes to the "vector_seq" file.

Quick Start

conda env create -f environment.yaml
conda activate dms_primer_design_env
python primer_design.py test_data/config.yaml

Config File Parameters

• output_prefix: (string) The name of the gene of interest
• output_path: (string) Directory where .tsv file will be saved
• rng_seed: (integer) Used to seed where synonymous and stop variants are designed
• wt_seq: (string) Path to .gb file containing the coding sequence for the gene of interest from which SNP-accesible variants are designed
• vector_seq: (string) Path to alternate .gb file containing the coding sequence for the gene of interest. Can be identical to the wt_seq path, but provides the option to design variants for a wildtype nucleotide sequence that may differ from a codon-optimized nucleotide sequence on the vector plasmid
• homology_length: (integer) Length of homology arms for Gibson Assembly of PCR fragments
• max_oligo_length: (integer) The ideal maximum length for each primer, but will extend if necessary to increase the melting temperature of the primer
• fwd_primer_melt_temp: (integer) Melting temperature of the forward doped primer that generates the variant
• rev_primer_melt_temp: (integer) Melting temperature of the reverse primer for a given tile
• mutagenesis_type: (string) Default is "missense" to generate only SNP-accessible variants, currently no option for saturation mutagenesis
• variant_windows: (list of strings) A list of feature names in the .gb file that specify where each tile of variants should be made
• synonymous_variant_rate: (floag) Value ranging from 0-1 denoting the percent of synonymous variants, e.g a value of .05 would result in 5% of the designed primers encoding synonymous variants, used to generate control variants
  remove_stop_variant_rate: (float) Value ranging from 0-1 denoting the percent of stop variants removed from primer design, e.g a value of 1.0 would result in all stop variants being excluded from the primer design, used to generate control variants