Safeport and recoding (SPaRC) project

This project aims to annotate genomic regions that are safe for gene editing (called safeports) and to to recode a genomic region by changing specific codons, e.g. replacing al GTA with GTT in the exons. Therefore, the script allows automated downloads of FASTA and gff from Ensemble via its REST API, annotates safeports, recodes the sequence and then creates a GenBank file. It mainly depends on BioPython for storing the sequence and annotation data.

Required modules

This code runs in Python 3. Please check requirements.txt for required modules and their versions.

Usage

The tool can be executed using the following command line structure:

python main.py region [-v] [-f flank] [-o dir] [-n prefix] [-p annotation] [-l] [-c
FILE] [-e length] [-sm {max,soft,maneonly}] [-j True]

positional arguments:

region either gene name/symbol or region of interest

optional arguments:

• -h, --help
  show this help message and exit
• -V, --version
  show program's version number and exit
• -v, --verbose
  Set logging level (default info)
• -f, --gene-flank
  Gene flanking region size (default 0)
• -o, --output-dir
  Output directory (default ./)
• -n, --output-prefix
  Output file prefix (default uses input region)
• -p, --safeport-filter
  List annotations that are NOT safeports (default 'ALL')
• -l, --safeport-length
  Minimum length for safeports (default 3nt)
• -c, --coding-file
  Optional codon mapping file
• -e, --endprotect
  Protect the end of each coding sequence by not modifying the last n nt
• -sm, --manestrength
  Choose 'max' (if none of the overlapping cds is a MANE transcript, the longest cds will be taken),'soft'(if none of the overlapping transcripts is a MANE, none will be recoded), or 'maneonly' (only MANE transcripts will be recoded).
• -j, --join
  If True, a joined CompoundLocation annotations will be created for each transcript.

Encoded presumptions

• Organism is Human, assembly "GRCh38"
• REST API
  • Included features in GFF3 download:\
    • feature=regulatory;feature=cds;feature=exon;feature=variation;feature=repeat;\
    • variant_set=1kg_3_com (Variants genotyped by the 1000 Genomes project (phase 3) with frequency of at least 1%)
  • MANE transcripts:
    • Use Ensembl ID for transcripts
• Safeports
  • Short names for list of annotations that are not region for safeports:
    • ALL: ['exon','CDS','CTCF_binding_site','promoter','sequence_variant','repeat_region','TF_binding_site']
    • noSNP: ['exon','CDS','CTCF_binding_site','promoter','repeat_region','TF_binding_site']
• Make GenBank file
  • Molecule type == 'DNA'
  • Taxonomy of Homo Sapiens

Workflow

Downloading the GFF3 and FASTA file of a region or gene of interest: ensemble_rest.py

The user can either input a gene name/symbol or a region of interest. The GFF3 and FASTA data for a specified region is accessed on https://rest.ensembl.org. The framework for reading the GFF3 file and converting it into a GenBank file is based on bcbio-gff (https://github.com/chapmanb/bcbb/blob/master/gff/Scripts/gff/gff_to_genbank.py).

Creating safeports: safeports.py

Safeports are created in regions that have no other relevant annotation via createSAFEPORTS. The default annotations taken into account for this are 'exon', 'CDS', 'CTCF_binding_site', 'promoter', 'sequence_variant’, 'TF_binding_site’ and ‘repeat_region'.

Recoding

The recode function replaces the codons based on codon_map in the sequence record, but protects nucleotides at the beginning or end of the coding sequence and handles overlaps based on a list of MANE transcripts. Even for coding sequences that are not overlapping, the user can choose to only recode MANE transcripts by indicating “maneonly”. Otherwise, if overlaps occur, the MANE transcript is recoded preferably. If the “soft” option is chosen, no coding sequence is recoded if the overlapping coding sequences are not part of a MANE transcript. If the “max” option is chosen, the longer coding sequence will be recoded, even if it’s not a MANE transcript.

License

All code, images and documents in this repository are freely available for all uses. Code is available under the MIT license and images, documentations and talks under the Creative Commons No Rights Reserved (CC0) license.