CRISPR-detector provides a web-hosted platform (https://db.cngb.org/crispr-detector/) and local deployable pipeline to fast and accurately identify and annotate editing-induced mutations from genome editing assays.
CRISPR-detector pipeline possesses 4 key innovations :
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optimized processing speed allowing for hundreds of amplicons and whole genome scanning;
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edited and control sample co-analysis, to remove background variants exist prior to genome editing;
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functional and clinical annotation of editing-induced mutations;
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integrated structural variation (SV) calling.
Download sentieon toolkit from
https://s3.amazonaws.com/sentieon-release/software/sentieon-genomics-202010.03.tar.gz
You may request a license by sending emails to huanglei@genomics.cn
export SENTIEON_LICENSE=PATH_TO_SENTIEON/sentieon-genomics-202010.03/localhost_eval.lic
export PATH=PATH_TO_SENTIEON/sentieon-genomics-202010.03/bin:$PATH
pip install biopython
pip install pyfaidx
pip install -U textwrap3
conda install blast
conda install samtools
Download ANNOVAR from https://www.openbioinformatics.org/annovar/annovar_download_form.php
perl annotate_variation.pl -downdb -webfrom annovar avdblist humandb/ -buildver hg38
perl annotate_variation.pl -buildver hg38 -downdb -webfrom annovar refGene humandb/
perl annotate_variation.pl -buildver hg38 -downdb -webfrom annovar clinvar_20210501 humandb/
export PATH=PATH_TO_ANNOVAR/annovar:$PATH
Organism Homo sapiens experiment type sequencing data support variant annotations from refGene & ClinVar, other species may only support refGene annotations
You may build ANNOVAR database youself for any species with coresponding genome assembly and gff3 format files
For example, to build a database for zebrafish. Download GRCz11.fa and GRCz11.gff3 from public database.
Then running commands as following:
conda install -c bioconda/label/cf201901 gffread
conda install -c bioconda/label/cf201901 ucsc-gtftogenepred
conda install -c bioconda/label/cf201901 blast
cd PATH_TO_ANNOVAR/
mkdir zebrafishdb && cd zebrafishdb
mv */GRCz11.fa zebrafishdb
mv */GRCz11.gff3 zebrafishdb
gffread GRCz11.gff3 -T -o GRCz11.gtf
gtfToGenePred -genePredExt GRCz11.gtf GRCz11_refGene.txt
retrieve_seq_from_fasta.pl --format refGene --seqfile GRCz11.fa GRCz11_refGene.txt --out GRCz11_refGeneMrna.fa
makeblastdb -in GRCz11.fa -dbtype nucl
python CRISPRdetectorCORE.py
--sample: sample name & output dir
--e1: treatment group fq1 path, required = True
--e2: treatment group fq2 path, required = False
--c1: control group fq2 path, required = False
--c2: control group fq2 path, required = False
--sample: sample name & output dir name, required = True
--amplicons_file: a tab-delimited text amplicons description file with up to 3 columns, required=True
[AMPLICON_NAME, AMPLICON_SEQ, gRNA_SEQ_without_PAM(Optional)]
--threads: number of threads to run sentieon minimap2 & driver module, default=1
--anno: annotate variants with ANNOVAR [1] or not run ANNOVAR [2], required = False
--assembly: assembly version, hg19,hg38 ...
--db: ANNOVAR database path, required = False
--ClinVar: only organism homo sapiens experiment type sequencing data support variant annotations from ClinVar[1], default = 0
--cleavage_offset: Center of quantification window to use within respect to the 3-end of the provided sgRNA sequence, default = -3
--window_size: defines the size (in bp) of the quantification window extending from the position specified by the cleavage_offset parameter in relation to the provided guide RNA sequence, 0 means whole amplicon analysis, default = 0
--o: output path, default='.', required = False
--ignore_substitutions: enable substitutions evaluation[1], default = 0
--min_tumor_allele_frac: the minimum allelic fraction in treated sample, default = 0.005
--min_num_of_reads: the minimum number of reads (per locus site) to evaluate, default = 500
--max_fisher_pv_active: the maximum pvalue of the statistical difference between treated and untreated sample, default = 0.05
python CRISPRdetectorWGS.py
--e1: treatment group fq1 path, required = True
--e2: treatment group fq2 path, required = False
--c1: control group fq2 path, required = False
--c2: control group fq2 path, required = False
--sample: sample name & output dir
--threads: number of threads to run sentieon minimap2 & driver module, default = 1
--bed: bed format file input to call variants of interest region, required = False
--anno: annotate variants with ANNOVAR [1] or not run ANNOVAR [2], required = False
--assembly: path to assembly in fasta format : hg38.fa mm9.fa ... required = False
--species: Homo_sapiens,Mus_musculus... required=False
--db: ANNOVAR database path, required=False