10. Examples

In all the following examples, the current directory/working directory is presumed to contain all files in https://github.com/rlrq/MINORg/tree/master/examples.

10.1. Example 1

Generate gRNA in the coding regions of a reference gene.

We will use the gene ‘AT1G33560’
As the default feature type for generating gRNA in is “CDS” (coding region), we do not need to explicitly specify it

Python code

>>> from minorg.MINORg import MINORg
>>> my_minorg = MINORg(prefix = "example1")
>>> my_minorg.add_reference("TAIR10", "subset_ref_TAIR10.fasta", "subset_ref_TAIR10.gff")
>>> my_minorg.genes = "AT1G33560"
>>> my_minorg.query_reference = True
>>> my_minorg.seq() ## generate target sequence(s), in this case that of AT1G33560
>>> my_minorg.grna() ## generate all possible gRNA in target sequence(s)
>>> my_minorg.filter_feature() ## filter gRNA by feature type
>>> my_minorg.write_pass_grna_fasta() ## write gRNA that pass all filters (in this case, only 1 filter was used)
>>> my_minorg.pass_fasta
'/path/to/current/directory/example1/example1_gRNA_pass.fasta'

CLI code (bash)

$ minorg --prefix example 1 \
         --assembly ./subset_ref_TAIR10.fasta --annotation ./subset_ref_TAIR10.gff \ ## reference genome
         --indv ref --gene AT1G33560 \ ## query genome and gene
         --gc-min 0 --gc-max 1 \ ## accept all gRNA regardless of GC content
         --auto ## auto-approve minimum gRNA sets

Note that the CLI code invokes the full programme, so minimum gRNA set(s) will also be generated. Nevertheless, you can still find a FASTA file of all passing gRNA (that are within the desired feature AND have desired GC content) at ‘/path/to/current/directory/example1/example1_gRNA_pass.fasta’.

CLI code (bash): using reference genome aliases

$ minorg --prefix example 1 \
         --reference-set ./arabidopsis_genomes.txt --ref TAIR10 \ ## reference genome
         --indv ref --gene AT1G33560 \ ## query genome and gene
         --gc-min 0 --gc-max 1 \ ## accept all gRNA regardless of GC content
         --auto ## auto-approve minimum gRNA sets

Here, we use --reference-set to give MINORg a mapping of reference genome FASTA and GFF files to more easy to write aliases. In this case, the alias for the combination of ‘./subset_ref_TAIR10.fasta’ and ‘./subset_ref_TAIR10.gff’ is ‘TAIR10’, which we pass to --ref.

CLI code (bash): using config.ini

Open ./sample_config.ini and replace ‘/path/to/arabidopsis_genomes.txt’ with the absolute path to ./araidopsis_genomes.txt. Save it.

$ export MINORG_CONFIG=./sample_config.ini
$ minorg --prefix example 1 \
         --indv ref --gene AT1G33560 \ ## query genome and gene
         --gc-min 0 --gc-max 1 \ ## accept all gRNA regardless of GC content
         --auto ## auto-approve minimum gRNA sets

If we open ‘./sample_config.ini’, we will find the following lines:

reference sets = athaliana:/path/to/athaliana_genomes.txt
                 arabidopsis:/<absolute path>/arabidopsis_genomes.txt

These lines tell MINORg to use ‘arabidopsis’ as an alias for arabidopsis_genomes.txt.

reference set = arabidopsis

This line tells MINORg to pass arabidopsis_genomes.txt (mapped from alias ‘arabidopsis’) to --reference-set.

reference = TAIR10

And finally this line tells MINORg to use the FASTA and GFF combination mapped to the alias ‘TAIR10’ in arabidopsis_genomes.txt as the reference genome.

10.2. Example 2

Description of desired outcome

Python CLI