Python script for generating oligo libraries and PCR primers for deep mutational scanning (DMS) of gene of interest. The mutation type for DMS library can be customized based on the personal needs which include single amino acid replacement, deletion and insertion. The oligo library design is meant to be compatible for Golden gate assembly using BsaI.
biopython
numpy
-> Targeted genes must be in fasta format and include a minimum of 30 bases surrounding gene.
-> Entire plasmid sequence with all the essential genetic context is advised to search for nonspecific amplification
-> Multiple sequences can be input for deep mutation scanning in one file.
-> Final output is fasta format. One file for oligo pools and one file for PCR primers
-> Different mutation type function can be run separately. The barcode might be overlapped if the functions are run separately.
-> "allmut" function wrapped up everything and generate oligo libraries based on the same fragmentation strategy of the GOI. And it generates all mutation types without the overlap of barcode usage.
-> Note: "allmut" and "allmut_noCys" functions generate different length of oligos, because all of the mutation types require to fit into same frame. Based on insertion, deletion and replacement, the length of oligo varies.
-> The primers for both of the oligo libraries or plasmid vector within the same fragmentation part are generic.
Gene primers should be same for the same sequence, because no matter what the mutation types we introduced here the fragmentation strategy should always be the same based on the size of the GOI
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Gene start is defined as the position / base number of the first base in the '-1 amino acid' of the first amino acid we want to mutagenize.
e.g. ATGACTGTACCGCAC
protein sequence : M T V P H
If the DMS starts from amino acid V, the start position should be base number of A in codon ACT (amino acid T) -
Gene end is defined as base number of the last base in the '+1 codon' of the last codon we want to mutagenize.
e.g. ATGACTGTACCGCAC
protein sequence : M T V P H
If the DMS ends at amino acid P, the end position should be the base number of the last C in codon CAC (amino acid H) (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'
-> Final version for running DMS: 'main_spine.py' with functional module 'SPINE_v7.py' SPINE_v7 contains all of the single amino acid mutation function and also the integrated version for bulk deep mutation scanning
DMS: single aa replacement
S_INS/S_DEL: single aa insertion / deletion
allmut: combination of all single aa mutation types
allmut_noCys: exclude introduction of Cysteine from the all mutation types
-> SPINE_v7 also allows for multiple input of GOI sequences in one input file
-> SPINE_v7 carries counter to generate the diversity of final oligo libraries
-> SPINE_BsaI is modified to generate DMS library with type II site BsaI. And it has been troubleshooted to fix the problem of introducing extra BsaI site after DMS.
-> Fasta_converter.ipynb is used to convert fasta file to csv file. And it contains a function to deduplicate the oligos by identifying BsaI as token and extract oligo sequence translating it into amino acid. The final deduplicating will be run based on the repeat of amino acid sequence. The duplicate is caused by insertion and deletion. (e.g. amino acid: NDK. Inserting D at the second and third position are the same -- NDDK. For deletion, if there are continuous same amino acids, such as NDDK, deletion of the second or the third amino acid generates same result )
Domain Insertion Scanning: Note: Domain Insertion scanning is directly from the original script. I did not make any changes.
python3.8 main_spine.py -wDir tests -geneFile combined_fasta.fa -oligoLen 230 -mutationType DIS
Deep Mutational Scanning:
python3.8 main_spine.py -wDir test_bulk_mut -geneFile Multiple_inputfile.fa -oligoLen 230 -mutationType allmut -usage ecoli
python3.8 main_spine.py -wDir test_bulk_mut -geneFile Kir.fa -oligoLen 230 -mutationType DMS -usage ecoli
optional arguments:
-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).
-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 could help with insertion coverage close to
fragment boundary. Overlap does not add additional
insertions and thus no additional oligos.
-mutationType Run deep insertion scan "DIS", deep mutation scan for single amino acid replacement "DMS", single amino acid deletion "S_DEL",
single amino acid insertion "S_INS" or combine all single amino acid mutation together(replacement, deletion, insertion) "allmut".
Or exclude any introduction of 'Cys' into the mutation library "allmut_noCys"
-usage USAGE Default is "human". Or select "ecoli"