ANGIE - ANalysis of GenomIc Excisions tiggered by CRISPR-Cas

ANGIE is an analytic workflow to investigate CRISPR-Cas-induced genomic excisions. This repository provides a set of scripts that are focused on analyzing CRISPR-Cas9 multiplexed plasmid libraries, which aim to excise genomic regions (preferably exons) of a specified gene. The excisions can be identified by extracting the mRNA. The mRNA needs to be converted to cDNA and sequenced using Oxford NanoPore. Additional to the NanoPore sequencing, the guides present in the cell need to be sequenced using NGS.

The repository provides a YAML file from which a conda environment can be created, which includes all dependencies. conda env create -f conda_env.yml

For more details, visit the !

Data

The final data needed to run this Pipeline is:

• A FASTQ-file containing all NanoPore reads
• The reference gene in FASTA format
• Exon gene annotation file in BED3 format
• All guide sequences in FASTA format
• A TSV-file with guide pair NGS counts
  • Example file:

    GuideA	GuideB	Rb_Tiling_ctrl_A	Rb_tiling_lib	Rb_Tiling_ctrl_C	Rb_Tiling_end_B	Rb_Tiling_end_D
    exon1_1234_0.60	exon2_1234_0.60	100	50	112	345	367
    exon1_1234_0.60	exon3_1234_0.60	20	25	10	490	510
    exon1_1234_0.60	exon4_1234_0.60	101	120	39	1123	1000

Note: The guide names need to be in following format: exon[X]_[locus]_[doench score]

Workflow

Output

Scatterplot

1 Scatterplot of Guide Exon-Exon pairs. Dot size indicates number of pairs. Color indicates log2fold change from library to end state. 2 Scatterplot of Excision pairs found in NanoPore reads. Size indicates normalized count. Color indicates mean excision length. 3 Scatterplot of Excision pairs not found in NanoPore reads. Color indicates mean excision length. Quadrants are indicated by named boxes A, B and C.

Quadrant stats

Percentage of found excisions compared to possible excisions per quadrant.

Boxplots of mean excision length and normalized NanoPore count per quadrant.