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README.md

MASCOT: Model-based Analysis of Single-cell CRISPR knockOuT screenin

  • MASCOT is the first one-step applicable pipeline based on topic model to analyze single-cell CRISPR screening data (independently termed Perturb-Seq, CRISP-seq, or CROP-seq), which could help to prioritize the knockout gene impact in a cellular heterogeneity level.

  • MASCOT is an integrated pipeline for model-based analysis of single cell CRISPR knockout screening data. MASCOT consists of three steps: data preprocessing, model building and perturbation effect prioritizing:

    • Data preprocessing: Besides the routine quality control and data normalization applied in single-cell RNA-seq analysis, MASCOT addresses several considerations that should be taken into account for such a novel data type: (1) Filtering cells with an extremely large proportion of zero knockout expression among the control cells, as these cells are inherently noisy or meaningless. (2) Reducing the false positive rate of gene knockout by considering sgRNA knockout efficiency; and (3) Filtering knockout cells without sufficient number to capture the corresponding perturbation phenotype.
    • Model building: MASCOT builds an analytical model based on Topic Models to handle single-cell CRISPR screening data. The concept of topic models was initially presented in machine learning community and has been successfully applied to gene expression data analysis. A key feature of topic model is that it allows each knockout sample to process a proportion of membership in each functional topic rather than to categorize the sample into a discrete cluster. Such a topic profile, which is derived from large-scale cell-to-cell different knockout samples, allows for a quantitative description of the biologic function of cells under specific gene knockout conditions. MASCOT addresses several specific issues when applying the topic model to this specific data type: (1) Differences between different overall knockout efficiencies(OKE) are considered and rectified in the evaluation of the perturbation effect on the corresponding cells; (2) The distribution of topics between cases and controls is affected by the ratio of their sample numbers, and such a sample imbalance issue is addressed when prioritizing the perturbation effect. (3) The optimal topic number is automatically selected by MASCOT in a data-driven manner.
    • Perturbation effect prioritizing: Based on the model-based perturbation analysis, MASCOT can quantitatively estimate and prioritize the individual gene knockout effect on cell phenotypes from two different perspectives, i.e., prioritizing the gene knockout effect as an overall perturbation effect, or in a functional topic-specific way.
  • Input File Format. For running MASCOT, the input data needed to follow the standard format we defined. For convenience, MASCOT accepts two kinds of input data formats: (1) The first data format can be referred in the data_format_example/crop_unstimulated.RData we provided. It is an example dataset containing "crop_unstimulated_expression_profile", "crop_unstimulated_sample_info_gene" and "crop_unstimulated_sample_info_sgRNA". You can apply function "Input_preprocess()" to handle this data format; (2) The second data format can be referred in the data_format_example/perturb_GSM2396857/ generated by 10X genomics. The directory data_format_example/perturb_GSM2396857 contains "barcodes.tsv", "genes.tsv", "matrix.mtx", "cbc_gbc_dict.tsv" and "cbc_gbc_dict_grna.tsv". You can apply function "Input_preprocess_10X()" to handle this data format.

  • Attention: (1) The label of the control sample needs to be "CTRL", and the name of the nontargeting sgRNA needs to be CTRL_xxx or xxx_CTRL_xxx; (2) The name of targeting sgRNA should like this: targetingGene_xxx or xxx_targetingGene_xxx. Although most single-cell CRISPR screening datasets are in accordance with our regulations, users are encouraged to check it beforehand.

  • For illustration purpose, we only took the unstimulated cell data applied in CROP-seq as an example.

    • Install: R>=3.4.1. You can install the MASCOT package from Github using devtools packages.
    require(devtools)
    install_github("BinDuan/MASCOT")
    library(MASCOT)
    # take crop_unstimulated.RData as an example.
    dim(crop_unstimulated_expression_profile)
    ## [1] 36722  2646
    
    crop_unstimulated_expression_profile[1:3,1:3]
    ##          GCAGTCCTTCTN ACGTAGGGGTAN AAACAACCGAAN
    ## A1BG                0            0            0
    ## A1BG-AS1            0            0            0
    ## A1CF                0            0            0
    
    # sample_info_gene.
    length(crop_unstimulated_sample_info_gene)
    ## [1] 2646
    
    class(crop_unstimulated_sample_info_gene)
    head(crop_unstimalated_sample_info_gene)
    ## [1] "character"
    
    ## GCAGTCCTTCTN ACGTAGGGGTAN AAACAACCGAAN TCAGTGGCTTCT AGTATTCTCACN TTATAGCATGCA 
    ##      "NR4A1"     "NFATC2"       "CTRL"       "CTRL"       "CTRL"       "CTRL"
    
    # sample_info_sgRNA.
    length(crop_unstimulated_sample_info_sgRNA)
    ## [1] 2646
    
    class(crop_unstimulated_sample_info_sgRNA)
    head(crop_unstimulated_sample_info_sgRNA)
    ##[1] "character"
    
    ##         GCAGTCCTTCTN          ACGTAGGGGTAN    AAACAACCGAAN   TCAGTGGCTTCT    AGTATTCTCACN    TTATAGCATGCA  
    ## "Tcrlibrary_NR4A1_1"  "Tcrlibrary_NFATC2_1"    "CTRL00698"    "CTRL00320"     "CTRL00087"     "CTRL00640" 
    
    • The first step: data preprocessing.
    # For "crop_unstimulated.Rdata", this function integrates the input data and filters mitochondrial ribosomal protein(^MRP) and ribosomal protein(^RP).
    crop_seq_list<-Input_preprocess(crop_unstimulated_expression_profile,crop_unstimulated_sample_info_gene,crop_unstimulated_sample_info_sgRNA,sample_info_batch=NULL)
    
    # For data format like "data_format_example/perturb_GSM2396857" generated by 10X genomics, function "Input_preprocess_10X()" will be suitable. Users can also change this data format to the standard format like "crop_unstimulated.Rdata", then use function "Input_preprocess()" to process it. 
    # cell quality control
    crop_seq_qc<-Cell_qc(crop_seq_list$expression_profile,crop_seq_list$sample_info_gene,gene_low=500,species="Hs",plot=T)

    # other filtering steps, including "nonzero_ratio", "sgRNA efficiency" and "phenotype capture".
    crop_seq_filtered<-Cell_filtering(crop_seq_qc$expression_profile_qc,crop_seq_qc$sample_info_gene_qc,crop_seq_list$sample_info_sgRNA,nonzero=0.01,grna_cell_num=10,fold_change=0.5,plot=T)

    # plot the overvieww of cell filterings.
    component<-Plot_filtering_overview(crop_seq_list$sample_info_gene,crop_seq_qc$sample_info_gene_qc,crop_seq_filtered$nonzeroRatio,crop_seq_filtered$sample_info_gene_qc_zr_se,crop_seq_filtered$sample_info_gene_qc_zr_se_pc)

    • The second step: model building
    # obtain highly dispersion differentially expressed genes.
    crop_seq_vargene<-Get_high_var_genes(crop_seq_filtered$expression_profile_qc_zr_se_pc,crop_seq_filtered$sample_info_gene_qc_zr_se_pc,plot=T)

    # get topics and select the optimal topic number automatically.
    optimal_topics<-Get_topics(crop_seq_vargene,crop_seq_filtered$sample_info_gene_qc_zr_se_pc,plot=T)

    # plot a heatmap to show the distribution of cells for each topic.
    Plot_cell_topic(optimal_topics)

    # annotate each topic's functions. Hs(homo sapiens) or Mm(mus musculus) are available.
    topic_func<-Topic_func_anno(optimal_topics,species="Hs",plot=T)

    # get off-target information. This step won't affect the final ranking result, but just present the off-target information. In most cases, the sgRNA has no off-targets. If you do not want to consider this factor, then just skip this step. 
    library(CRISPRseek)
    library("BSgenome.Hsapiens.UCSC.hg38")
    library(TxDb.Hsapiens.UCSC.hg38.knownGene)
    library(org.Hs.eg.db)
    library(org.Mm.eg.db)
    gRNAFilePath<-"extdata/crop_unstimulated_sgrna.fa"
    crop_results <- offTargetAnalysis(inputFilePath = gRNAFilePath, findgRNAs = FALSE,findgRNAsWithREcutOnly = FALSE,findPairedgRNAOnly = FALSE, BSgenomeName = Hsapiens,txdb = TxDb.Hsapiens.UCSC.hg38.knownGene,min.score=1,scoring.method = "CFDscore",orgAnn = org.Hs.egSYMBOL, max.mismatch = 3,outputDir=getwd(), overwrite = TRUE)
    # then, check if there are off-targets.
    offTarget_Info<-Get_offtarget(crop_results,crop_seq_filtered$expression_profile_qc_zr_se_pc,crop_seq_filtered$sample_info_gene_qc_zr_se_pc,crop_seq_list$sample_info_sgRNA)
    
    • The third step: perturbation effect prioritizing
    # calculate topic distribution for each cell.
    distri_Diff<-Get_distribution_diff(optimal_topics,crop_seq_filtered$sample_info_gene_qc_zr_se_pc,crop_seq_filtered$KO_efficiency)
    
    # calculate the overall perturbation effect ranking list without "offTarget_Info" calculated.
    rank_overall_result<-Rank_overall(distri_Diff)
    #rank_overall_result<-Rank_overall(distri_Diff,offTarget_hash=offTarget_Info) (if "offTarget_Info" was calculated).
    
    # calculate the topic-specific ranking list.
    rank_topic_specific_result<-Rank_topic_specific(rank_overall_result$rank_overall_result_detail)
    
    • output
    # output the overall perturbation effect ranking list with a summary or detailed style.
    write.table(rank_overall_result$rank_overall_result_summary,"~/rank_overall_result_summary.txt",col.names=T,row.names=F,quote=F,sep="\t")
    write.table(rank_overall_result$rank_overall_result_detail,"~/rank_overall_result_detail.txt",col.names=T,row.names=F,quote=F,sep="\t")
    
    # output the topic-specific ranking list with a summary or detailed style.
    write.table(rank_topic_specific_result$rank_topic_specific_result_summary,"~/rank_topic_specific_result_summary.txt",col.names=T,row.names=F,quote=F,sep="\t")
    write.table(rank_topic_specific_result$rank_topic_specific_result_detail,"~/rank_topic_specific_result_detail.txt",col.names=T,row.names=F,quote=F,sep="\t")