# scMAGeCK

#### Wei Li, Xiaolong Cheng

#### Oct 22, 2019

## Introduction

scMAGeCK is a computational model to identify genes associated with multiple expression phenotypes from CRISPR screening coupled with single-cell RNA sequencing data (CROP-seq).

scMAGeCK is based on our previous MAGeCK and MAGeCK-VISPR models for pooled CRISPR screens, but further extends to scRNA-seq as the readout of the screening experiment. scMAGeCK consists of two modules: scMAGeCK-Robust Rank Aggregation (RRA), a sensitive and precise algorithm to detect genes whose perturbation links to one single marker expression; and scMAGeCK-LR, a linear-regression based approach that unravels the perturbation effects on thousands of gene expressions, especially from cells undergo multiple perturbations.

## Usage

### scmageck_rra

        library(scMAGeCK)
        ### BARCODE file contains cell identity information, generated from the cell identity collection step
        BARCODE <- system.file("extdata","barcode_rec.txt",package = "scMAGeCK")

        ### RDS can be a Seurat object or local RDS file path that contains the scRNA-seq dataset
        RDS <- system.file("extdata","singles_dox_mki67_v3.RDS",package = "scMAGeCK")

        ### Set RRA executable file path.
        ###    You can generate RRA executable file by following commands:
        ###        wget https://bitbucket.org/weililab/scmageck/downloads/RRA_0.5.9.zip
        ###        unzip RRA_0.5.9.zip
        ###        cd RRA_0.5.9
        ###        make
        RRAPATH <- "/Library/RRA_0.5.9/bin/RRA"

        target_gene <- "MKI67"

        rra_result <- scmageck_rra(BARCODE=BARCODE, RDS=RDS, GENE=target_gene, RRAPATH=RRAPATH,
                 LABEL='dox_mki67', NEGCTRL=NULL, KEEPTMP=FALSE, PATHWAY=FALSE, SAVEPATH=NULL)

    ## Checking RRA...

    ## RRA is not does not exist! Please check RRA executable file path

### scmageck_lr

        library(scMAGeCK)
        ### BARCODE file contains cell identity information, generated from the cell identity collection step
        BARCODE <- system.file("extdata","barcode_rec.txt",package = "scMAGeCK")
        ### RDS can be a Seurat object or local RDS file path that contains the scRNA-seq dataset
        RDS <- system.file("extdata","singles_dox_mki67_v3.RDS",package = "scMAGeCK")

        lr_result <- scmageck_lr(BARCODE=BARCODE, RDS=RDS, LABEL='dox_scmageck_lr',
                NEGCTRL = 'NonTargetingControlGuideForHuman', PERMUTATION = 1000, SAVEPATH=NULL, LAMBDA=0.01)

    ## Total barcode records: 8425

    ## Neg Ctrl guide: NonTargetingControlGuideForHuman

    ## Reading RDS file: D:/biocbuild/bbs-3.13-bioc/tmpdir/RtmpOs2LtK/Rinst3fe41b1c6244/scMAGeCK/extdata/singles_dox_mki67_v3.RDS

    ## Cell names in expression matrix and barcode file do not match. Try to remove possible trailing "-1"s...

    ## 6704 ...

    ## 6229 ...

    ## Index matrix dimension: 5698 , 30

    ## Selected genes: 25

    ## Permutation: 100 / 1000 ...

    ## Permutation: 200 / 1000 ...

    ## Permutation: 300 / 1000 ...

    ## Permutation: 400 / 1000 ...

    ## Permutation: 500 / 1000 ...

    ## Permutation: 600 / 1000 ...

    ## Permutation: 700 / 1000 ...

    ## Permutation: 800 / 1000 ...

    ## Permutation: 900 / 1000 ...

    ## Permutation: 1000 / 1000 ...

        lr_score <- lr_result[1]
        lr_score_pval <- lr_result[2]

## Output

### scmageck_rra

The scmageck_rra function will output the ranking and p values of each perturbed genes, using the RRA program in MAGeCK. Users familiar with the MAGeCK program may find it similar with the gene_summary output in MAGeCK.

Here is the example output of scMAGeCK-RRA:

    Row.names  items_in_group.low  lo_value.low  p.low  FDR.low goodsgrna.low  items_in_group.high  lo_value.high  p.high  FDR.high  goodsgrna.high
    TP53    271     0.11832 0.95619 1       48      271     1.014e-83       4.9975e-06      0.00015 184

Explanations of each column are below:

Column | Content
--- | ---
Row.names | Perturbed gene name
items_in_group.low | The number of single-cells with each gene perturbed
lo_value.low | The RRA score in negative selection (reducing the marker expression if this gene is perturbed). The RRA score uses a p value from rank order statistics to measure the degree of selection; the smaller score, the stronger the selection is. More information on the calculation of RRA score can be found in our original MAGeCK paper.
p.low | The raw p-value (using permutation) of this gene in negative selection
FDR.low | The false discovery rate of this gene in negative selection
goodsgrna.low | The number of single-cells that passes the threshold and is considered in the RRA score calculation in negative selection
items_in_group.high | The same as items_in_group.low: the number of single-cells with each gene perturbed)
lo_value.high | The RRA score in positive selection (increasing the marker expression if this gene is perturbed
p.high | The raw p-value (using permutation) of this gene in positive selection
FDR.high | The false discovery rate of this gene in positive selection
goodsgrna.high | The number of single-cells that passes the threshold and is considered in the RRA score calculation in positive selection

### scmageck_lr

The scmageck_lr function will generate several files below:

File | Description
--- | ---
lr_score | The score (similar with log fold change) of each perturbed gene (rows) on each marker gene (columns)
lr_score.pval | The associated p values of each score
LR.RData | An R object to store scores and p values

The format of score.txt and score.pval.txt is a simple table file with rows corresponding to perturbed genes and columns corresponding to marker genes. For example in the score.txt,

    Perturbedgene  APC                ARID1A               TP53               MKI67
         APC       0.138075836476524  -0.0343441660045313  0.214449590551132  -0.150287676553705

This row records the effects of perturbing APC gene on the expressions of APC, ARID1A, TP53 and MKI67.

## Contact us

Questions? Comments? Join the MAGeCK Google group or email us (wli2@childrensnational.org) directly.

Any advice and suggestions will be greatly appreciated.