Python script for generating oligo libraries and PCR primers for programmed domain insertion
Paper is linked here: (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-023-02880-6)
and here: (https://www.biorxiv.org/content/10.1101/2022.07.26.501589v1.full.pdf)
This code is licensed under the terms of the MIT license: License
Use Google Colab without downloading or installing on your computer
Run with a command line in terminal with the listed options below
Run with the GUI that uses tkinter
Targeted genes must be in fasta format and include the entire plasmid sequence to search for nonspecific amplification
Final output is fasta format. One file for oligo pools and one file for PCR primers
Gene start is defined as base number of first base in first codon and gene end is defined as base number of last base in last codon. (Program will subtract 1 from gene start for python numbering)
To define gene position within given fasta file, add start:# end:# to fasta description. (Otherwise use command line prompts)
'>geneA start:11 end:40'
Deep Mutational Scanning with amino acid subsitution with stop codons and ecoli codon usage, GGG insertion, 3 base deletion, and BsaI golden gate:
python run_dimple.py -wDir tests -geneFile combined_fasta.fa -oligoLen 230 -DMS -usage ecoli -include_stop_codons -restriction_sequence GGTCTC(G)1/5 -avoid_sequence GGTCTC -insertions GGG -deletions 3
options:
-h, --help show this help message and exit
-wDir WDIR Working directory for fasta files and output folder
-geneFile GENEFILE Input all gene sequences including backbone in a fasta format. Place all in one fasta file.
Name description can include start and end points (>gene1 start:1 end:2)
-handle HANDLE Genetic handle for domain insertion. This is important for defining the linker. Currently
uses BsaI (4 base overhang), but this can be swapped for SapI (3 base overhang).
-dis DIS use the handle to insert domains at every position in POI
-matchSequences Find similar sequences between genes to avoid printing the same oligos multiple times.
Default: No matching
-oligoLen OLIGOLEN Synthesized oligo length
-fragmentLen FRAGMENTLEN
Maximum length of gene fragment
-overlap OVERLAP Enter number of bases to extend each fragment for overlap. This will help with insertions
close to fragment boundary
-DMS Choose if you will run deep deep mutation scan
-custom_mutations CUSTOM_MUTATIONS
Path to file that includes custom mutations with the format position:AA
-usage USAGE Default is "human". Or select "ecoli. Or change code"
-insertions INSERTIONS [INSERTIONS ...]
Enter a list of insertions (nucleotides) to make at every position. Note, you should enter
multiples of 3 nucleotides to maintain reading frame
-deletions DELETIONS [DELETIONS ...]
Enter a list of deletions (number of nucleotides) to symmetrically delete (it will make
deletions in multiples of 2x). Note you should enter multiples of 3 to maintain reading
frame
-include_substitutions INCLUDE_SUBSTITUTIONS
If you are running DMS but only want to insert or delete AA
-barcode_start BARCODE_START
To run DIMPLE multiple times, you will need to avoid using the same barcodes. This allows
you to start at a different barcode.
-restriction_sequence RESTRICTION_SEQUENCE
Recommended using BsmBI - CGTCTC(G)1/5 or BsaI - GGTCTC(G)1/5
-avoid_sequence AVOID_SEQUENCE [AVOID_SEQUENCE ...]
Avoid these sequences in the backbone - BsaI and BsmBI. For multiple sequnces use a space
between inputs. Example -avoid_sequence CGTCTC GGTCTC
-include_stop_codons Include stop codons in the list of scanning mutations.
-include_synonymous Include synonymous codons in the list of scanning mutations.
-make_double Make each combination of mutations within a fragment
Run the GUI with the commandline prompt: python run_dimple_gui.py
You must select:
-Target Gene File - select fasta file with name description including start and end points (>gene1 start:1 end:2)
-One or more of the mutations to make to the target gene
Run the program by pressing Run DIMPLE