/MAGeCKFlute

MAGeCKFlute

1. sgRNA count by MAGeCK v2.0.5

mageck count --trim-5 1 -l CRISPR_sgRNA_library.csv -n Cal27_NSD1KO --sample-label Cal27_day0,Cal27_day14,Cal27_NSD1KO_day0,Cal27_NSD1KO_day14  --fastq Cal27_day0.fastq Cal27_day14.fastq Cal27_NSD1KO_day0.fastq Cal27_NSD1KO_day14.fastq

2. Enrichment analysis using MAGeCK mle module

mageck mle -k Cal27_NSD1KO.count_normalized.txt -d designmat.txt -n Cal27_NSD1KO_mle_normalized

3. Integrative analysis pipeline for pooled CRISPR functional genetic screens - MAGeCKFlute

################### Run following codes in R #################
# source("http://www.bioconductor.org/biocLite.R")
# biocLite("MAGeCKFlute")
library(MAGeCKFlute)
## Load gene summary data in MAGeCK MLE results
mle.gene_summary = read.delim("Cal27_NSD1KO_mle_normalized.gene_summary.txt", header=T, sep="\t")
## Run the downstream analysis pipeline for MAGeCK MLE
FluteMLE(mle.gene_summary, ctrlname="Cal27_day14", treatname="Cal27_NSD1KO_day14", prefix="MLE", organism="hsa")
###############################################################

4. Quality control

################### Run following codes in R #################
library(MAGeCKFlute)
## Load gene summary data in MAGeCK MLE results
countsummary = read.delim("Cal27_NSD1KO.countsummary.txt", header=T, sep="\t")
## View mapping ratio of each sample
MapRatesView(countsummary)
## View evenness of sgRNA reads in each sample
IdentBarView(countsummary, x = "Label", y = "GiniIndex", ylab = "Gini index", main = "Evenness of sgRNA reads")
## View number of missed sgRNAs in each sample
countsummary$Missed = log10(countsummary$Zerocounts)
IdentBarView(countsummary, x = "Label", y = "Missed", fill = "#394E80", ylab = "Log10 missed gRNAs", main = "Missed sgRNAs")
###############################################################