###Workflow for generating Figure 4 of "CRISPR MultiTargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences" manuscript

Step 1. Making a list of alternative transcripts.

This step makes a list of all alternative transcripts in zebrafish together with their corresponding genes based on the data available in Ensembl BioMart database. The genes_selecter.py script when run on zebrafish_transcript_table.txt accomplishes this task and produces genes_with_alternative_transcripts.txt file as output. However, Github has restrictions on the file sizes so the entire file was not possible to upload. We therefore opted to include zebrafish_transcript_table_example.txt file which has a similar structure. The complete file can be generated using BioMart.

Step 2. Finding unique sites in alternative transcripts and counting them.

For this step you need to run transcripts_cmdline.py on the zebrafish.db SQLite3 database using the genes_with_alternative_transcripts.txt file as input. The database was too big to include into this repository but its construction is described in the Database construction section. This script is designed to run on Windows using the Windows version of the ClustalW2 executable program. To run it, you will need to install ClustalW2 and configure the clustalw_exe variable, for example: clustalw_exe = r"C:\Program Files\ClustalW2\clustalw2.exe".

The output from this step are two files: total_unique_sites.txt and detailed_unique_sites.txt. The total_unique_sites.txt contains total counts of unique CRISPR/Cas9 sgRNA sites for all alternative transcripts analyzed in this workflow. The detailed_unique_sites.txt file contains the gene and transcript Ensemble identifiers of these alternative transcripts as well as counts of unique sgRNA sites in both sense and anti-sense strands of these transcripts, for example ENSDARG00000091715 ENSDART00000152509 14 6.

Step 3. Generating data for unique sgRNA target site overall statistics and making the Figure 4A plot

The proportions of "Genes with isoform-specific sgRNA sites", "Transcripts with isoform-specific sgRNA sites", "Sites in the sense strand", "Sites in the anti-sense strand" were calculated based on the detailed_unique_sites.txt file in Excel and the resulting values were pasted into the Figure4A.R R script.

Figure4A.R script was run to produce the plot in Figure 4A. Please see the Figure 4 included in this repository.

Step 4. Plotting the distribution of unique sgRNA counts in alternative transcripts for the Figure 4B.

Figure4B.R script was run with the total_unique_sites.txt file to produce the plot in Figure 4B. Please see the Figure 4 included in this repository.