Contents

Citation: if you use MAGeCKFlute in published research, please cite: Binbin Wang, Mei Wang, Wubing Zhang. “Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.” Nature Protocols (2019), doi: 10.1038/s41596-018-0113-7.t

1 Input data - weighted gene list

library(MAGeCKFlute)
file1 = file.path(system.file("extdata", package = "MAGeCKFlute"),
                  "testdata/rra.gene_summary.txt")
gdata = ReadRRA(file1)
genelist = gdata$Score
names(genelist) = gdata$id
genelist[1:5]
##   CREBBP    EP300      CHD C16orf72   CACNB2 
##  0.96608  1.02780  0.59265  0.82307  0.39268

2 Enrichment analysis methods

MAGeCKFlute incorporates three enrichment methods, including Over-Representation Test (ORT), Gene Set Enrichment Analysis (GSEA), and Hypergeometric test (HGT). Here, ORT and GSEA are borrowed from R package clusterProfiler (Yu et al. 2012).

2.1 Hypergeometric test

# Alternative functions EnrichAnalyzer and enrich.HGT.
hgtRes1 = EnrichAnalyzer(genelist[genelist< -1], method = "HGT")
head(hgtRes1@result)
##                                ID
## REACTOME_2467813 REACTOME_2467813
## REACTOME_72163     REACTOME_72163
## GO:0006364             GO:0006364
## GO:1901990             GO:1901990
## GO:0031145             GO:0031145
## REACTOME_68949     REACTOME_68949
##                                                             Description
## REACTOME_2467813                        Separation of Sister Chromatids
## REACTOME_72163                            mRNA Splicing - Major Pathway
## GO:0006364                                              rRNA processing
## GO:1901990            regulation of mitotic cell cycle phase transition
## GO:0031145       anaphase-promoting complex-dependent catabolic process
## REACTOME_68949                              Orc1 removal from chromatin
##                         NES       pvalue     p.adjust GeneRatio   BgRatio
## REACTOME_2467813 -10.350541 1.109937e-14 2.239853e-11    24/292 191/16544
## REACTOME_72163   -10.123151 3.683095e-14 3.716243e-11    23/292 183/16544
## GO:0006364        -8.586255 1.556246e-12 1.046835e-09    19/292 143/16544
## GO:1901990        -7.571253 1.344431e-11 6.782654e-09    14/292  80/16544
## GO:0031145        -7.591227 2.346509e-11 9.470511e-09    14/292  83/16544
## REACTOME_68949    -7.619360 3.195559e-11 1.074773e-08    13/292  71/16544
##                                                                                                                                          geneID
## REACTOME_2467813 5518/5686/5688/5689/5691/5692/5695/5702/5708/5347/29945/5885/701/8243/8697/9184/1778/10403/11243/147841/25936/54820/81930/9212
## REACTOME_72163   9775/1479/29894/23517/25804/5433/5435/10262/10523/10772/1665/23020/27339/3192/51340/56949/6426/6632/8175/1660/23398/54883/7536
## GO:0006364                          2091/23160/51367/55127/92856/6187/6209/9775/23517/5394/54512/56915/10969/23481/54555/81887/9136/54853/55661
## GO:1901990                                                                2068/5686/5688/5689/5691/5692/5695/5702/5708/5347/29945/701/8697/9184
## GO:0031145                                                                5686/5688/5689/5691/5692/5695/5702/5708/5347/29945/701/8697/9184/9212
## REACTOME_68949                                                                 4171/4175/5686/5688/5689/5691/5692/5695/5702/5708/9978/23594/890
##                                                                                                                                                          geneName
## REACTOME_2467813  PPP2R1A/PSMA5/PSMA7/PSMB1/PSMB3/PSMB4/PSMB7/PSMC3/PSMD2/PLK1/ANAPC4/RAD21/BUB1B/SMC1A/CDC23/BUB3/DYNC1H1/NDC80/PMF1/SPC24/NSL1/NDE/KIF18A/AURKB
## REACTOME_72163   EIF4A3/CSTF3/CPSF1/SKIV2L2/LSM4/POLR2D/POLR2F/SF3B4/CHERP/SRSF10/DHX15/SNRNP200/PRPF19/HNRNPU/CRNKL1/XAB2/SRSF1/SNRPD/SF3A2/DHX9/PPWD1/CWC25/SF1
## GO:0006364                                  FBL/WDR43/POP5/HEATR1/IMP4/RPS2/RPS15/EIF4A3/SKIV2L2/EXOSC10/EXOSC4/EXOSC5/EBNA1BP2/PES1/DDX49/LAS1L/RRP9/WDR55/DDX27
## GO:1901990                                                                     ERCC2/PSMA5/PSMA7/PSMB1/PSMB3/PSMB4/PSMB7/PSMC3/PSMD2/PLK1/ANAPC4/BUB1B/CDC23/BUB3
## GO:0031145                                                                     PSMA5/PSMA7/PSMB1/PSMB3/PSMB4/PSMB7/PSMC3/PSMD2/PLK1/ANAPC4/BUB1B/CDC23/BUB3/AURKB
## REACTOME_68949                                                                          MCM2/MCM6/PSMA5/PSMA7/PSMB1/PSMB3/PSMB4/PSMB7/PSMC3/PSMD2/RBX1/ORC6/CCNA2
##                  Count
## REACTOME_2467813    24
## REACTOME_72163      23
## GO:0006364          19
## GO:1901990          14
## GO:0031145          14
## REACTOME_68949      13
# hgtRes2 = enrich.HGT(genelist[genelist< -1])
# head(hgtRes2@result)

2.2 Over-representation test

# Alternative functions EnrichAnalyzer and enrich.ORT.
ortRes1 = EnrichAnalyzer(genelist[genelist< -1], method = "ORT")
head(ortRes1@result)
##                                ID
## REACTOME_2467813 REACTOME_2467813
## REACTOME_72163     REACTOME_72163
## GO:0006364             GO:0006364
## GO:1901990             GO:1901990
## GO:0031145             GO:0031145
## REACTOME_68949     REACTOME_68949
##                                                             Description
## REACTOME_2467813                        Separation of Sister Chromatids
## REACTOME_72163                            mRNA Splicing - Major Pathway
## GO:0006364                                              rRNA processing
## GO:1901990            regulation of mitotic cell cycle phase transition
## GO:0031145       anaphase-promoting complex-dependent catabolic process
## REACTOME_68949                              Orc1 removal from chromatin
##                         NES       pvalue     p.adjust GeneRatio   BgRatio
## REACTOME_2467813 -10.350541 4.600475e-14 9.283758e-11    24/292 191/16544
## REACTOME_72163   -10.123151 1.575004e-13 1.589179e-10    23/292 183/16544
## GO:0006364        -8.586255 7.998992e-12 5.380656e-09    19/292 143/16544
## GO:1901990        -7.571253 1.107947e-10 5.589591e-08    14/292  80/16544
## GO:0031145        -7.591227 1.853006e-10 7.478733e-08    14/292  83/16544
## REACTOME_68949    -7.619360 2.875071e-10 9.669822e-08    13/292  71/16544
##                                                                                                                                          geneID
## REACTOME_2467813 29945/5708/54820/5689/5695/5518/701/25936/5688/8697/81930/5702/5692/5347/10403/5691/147841/9212/11243/5885/1778/8243/5686/9184
## REACTOME_72163   6426/1479/56949/5435/6632/27339/23398/7536/54883/29894/5433/51340/10523/8175/10262/9775/1660/1665/3192/25804/10772/23020/23517
## GO:0006364                          23481/10969/2091/54555/23160/9136/51367/6187/55661/55127/6209/56915/54512/9775/54853/81887/5394/92856/23517
## GO:1901990                                                                2068/29945/5708/5689/5695/701/5688/8697/5702/5692/5347/5691/5686/9184
## GO:0031145                                                                29945/5708/5689/5695/701/5688/8697/5702/5692/5347/5691/9212/5686/9184
## REACTOME_68949                                                                 5708/5689/5695/5688/4175/5702/5692/5691/4171/890/9978/5686/23594
##                                                                                                                                                          geneName
## REACTOME_2467813 ANAPC4/PSMD2/NDE1/PSMB1/PSMB7/PPP2R1A/BUB1B/NSL1/PSMA7/CDC23/KIF18A/PSMC3/PSMB4/PLK1/NDC80/PSMB3/SPC24/AURKB/PMF1/RAD21/DYNC1H1/SMC1A/PSMA5/BUB3
## REACTOME_72163    SRSF1/CSTF3/XAB2/POLR2F/SNRPD1/PRPF19/PPWD1/SF1/CWC25/CPSF1/POLR2D/CRNKL1/CHERP/SF3A2/SF3B4/EIF4A3/DHX9/DHX15/HNRNPU/LSM4/SRSF10/SNRNP200/MTREX
## GO:0006364                                    PES1/EBNA1BP2/FBL/DDX49/WDR43/RRP9/POP5/RPS2/DDX27/HEATR1/RPS15/EXOSC5/EXOSC4/EIF4A3/WDR55/LAS1L/EXOSC10/IMP4/MTREX
## GO:1901990                                                                     ERCC2/ANAPC4/PSMD2/PSMB1/PSMB7/BUB1B/PSMA7/CDC23/PSMC3/PSMB4/PLK1/PSMB3/PSMA5/BUB3
## GO:0031145                                                                     ANAPC4/PSMD2/PSMB1/PSMB7/BUB1B/PSMA7/CDC23/PSMC3/PSMB4/PLK1/PSMB3/AURKB/PSMA5/BUB3
## REACTOME_68949                                                                          PSMD2/PSMB1/PSMB7/PSMA7/MCM6/PSMC3/PSMB4/PSMB3/MCM2/CCNA2/RBX1/PSMA5/ORC6
##                  Count
## REACTOME_2467813    24
## REACTOME_72163      23
## GO:0006364          19
## GO:1901990          14
## GO:0031145          14
## REACTOME_68949      13
# ortRes2 = enrich.ORT(genelist[genelist< -1])
# head(ortRes2@result)

2.3 Gene set enrichment analysis

# Alternative functions EnrichAnalyzer and enrich.GSE.
gseRes1 = EnrichAnalyzer(genelist, method = "GSEA")
## Warning in preparePathwaysAndStats(pathways, stats, minSize, maxSize, gseaParam, : There are ties in the preranked stats (2.63% of the list).
## The order of those tied genes will be arbitrary, which may produce unexpected results.
head(gseRes1@result)
##                                ID                             Description
## REACTOME_2467813 REACTOME_2467813         Separation of Sister Chromatids
## REACTOME_72163     REACTOME_72163           mRNA Splicing - Major Pathway
## GO:0006364             GO:0006364                         rRNA processing
## REACTOME_2500257 REACTOME_2500257 Resolution of Sister Chromatid Cohesion
## KEGG_hsa03013       KEGG_hsa03013                           RNA transport
## KEGG_hsa03040       KEGG_hsa03040                             Spliceosome
##                        NES       pvalue     p.adjust
## REACTOME_2467813 -2.346482 1.382185e-17 1.240925e-13
## REACTOME_72163   -2.299303 6.817860e-16 3.060537e-12
## GO:0006364       -2.243248 1.493703e-12 3.635892e-09
## REACTOME_2500257 -2.228855 1.619912e-12 3.635892e-09
## KEGG_hsa03013    -2.216234 4.329000e-12 7.773153e-09
## KEGG_hsa03040    -2.202327 1.166840e-11 1.745982e-08
##                                                                                                                                                                                                                                                                                  geneID
## REACTOME_2467813                    5719/23244/9735/11130/54821/55746/5709/79019/6396/23047/10197/1062/1063/57405/3796/113130/10274/5690/11004/9861/5905/701/5518/5702/8697/54820/9184/5686/81930/5885/29945/1778/5692/5695/5708/147841/25936/5688/8243/9212/10403/5689/5347/11243/5691
## REACTOME_72163   8449/6432/1655/9410/9092/25949/22827/55749/79869/1994/6628/6434/10450/23524/5432/5356/9939/9129/9785/51690/1477/10465/24148/26121/10250/8175/23398/51340/54883/5435/10262/10523/6426/9775/29894/1665/23020/1660/23517/25804/27339/7536/10772/1479/3192/5433/6632/56949
## GO:0006364                                                             79863/23246/317781/51202/79039/705/28987/55759/27042/29960/88745/22984/51118/10171/79707/10521/65083/54555/56915/6209/23160/2091/55661/9775/10969/5394/6187/51367/23481/54853/23517/92856/54512/9136/55127/81887
## REACTOME_2500257                                                                                                 23244/9735/11130/54821/55746/79019/6396/23047/1062/1063/57405/3796/113130/10274/11004/5905/701/5518/54820/9184/81930/5885/1778/147841/25936/8243/9212/10403/5347/11243
## KEGG_hsa03013                                                                                                               9972/55746/11097/6396/2733/9669/9939/25929/1975/11218/5976/10799/8891/5905/10250/9984/79897/9775/8892/51367/80145/8890/3837/60528/79833/8661/1983/1964/4927
## KEGG_hsa03040                                                                      8449/6432/1655/9410/9092/25949/22827/153527/8559/6628/6434/10450/5356/9939/9129/9785/51690/10465/24148/26121/8175/51340/10262/10523/6426/9984/9775/1665/23020/1659/25804/27339/10772/3192/6632/56949
##                                                                                                                                                                                                                                                                                                               geneName
## REACTOME_2467813                   PSMD13/PDS5A/KNTC1/ZWINT/ERCC6L/NUP133/PSMD3/CENPM/SEC13/PDS5B/PSME3/CENPE/CENPF/SPC25/KIF2A/CDCA5/STAG1/PSMB2/KIF2C/PSMD6/RANGAP1/BUB1B/PPP2R1A/PSMC3/CDC23/NDE1/BUB3/PSMA5/KIF18A/RAD21/ANAPC4/DYNC1H1/PSMB4/PSMB7/PSMD2/SPC24/NSL1/PSMA7/SMC1A/AURKB/NDC80/PSMB1/PLK1/PMF1/PSMB3
## REACTOME_72163   DHX16/SRSF7/DDX5/SNRNP40/SART1/SYF2/PUF60/CCAR1/CPSF7/ELAVL1/SNRPB/TRA2B/PPIE/SRRM2/POLR2C/PLRG1/RBM8A/PRPF3/DHX38/LSM7/CSTF1/PPIH/PRPF6/PRPF31/SRRM1/SF3A2/PPWD1/CRNKL1/CWC25/POLR2F/SF3B4/CHERP/SRSF1/EIF4A3/CPSF1/DHX15/SNRNP200/DHX9/MTREX/LSM4/PRPF19/SF1/SRSF10/CSTF3/HNRNPU/POLR2D/SNRPD1/XAB2
## GO:0006364                                                                                          RBFA/BOP1/DDX51/DDX47/DDX54/BYSL/NOB1/WDR12/UTP25/MRM2/RRP36/PDCD11/UTP11/RCL1/NOL9/DDX17/NOL6/DDX49/EXOSC5/RPS15/WDR43/FBL/DDX27/EIF4A3/EBNA1BP2/EXOSC10/RPS2/POP5/PES1/WDR55/MTREX/IMP4/EXOSC4/RRP9/HEATR1/LAS1L
## REACTOME_2500257                                                                                                               PDS5A/KNTC1/ZWINT/ERCC6L/NUP133/CENPM/SEC13/PDS5B/CENPE/CENPF/SPC25/KIF2A/CDCA5/STAG1/KIF2C/RANGAP1/BUB1B/PPP2R1A/NDE1/BUB3/KIF18A/RAD21/DYNC1H1/SPC24/NSL1/SMC1A/AURKB/NDC80/PLK1/PMF1
## KEGG_hsa03013                                                                                                                     NUP153/NUP133/NUP42/SEC13/GLE1/EIF5B/RBM8A/GEMIN5/EIF4B/DDX20/UPF1/RPP40/EIF2B3/RANGAP1/SRRM1/THOC1/RPP21/EIF4A3/EIF2B2/POP5/THOC7/EIF2B4/KPNB1/ELAC2/GEMIN6/EIF3A/EIF5/EIF1AX/NUP88
## KEGG_hsa03040                                                                             DHX16/SRSF7/DDX5/SNRNP40/SART1/SYF2/PUF60/ZMAT2/PRPF18/SNRPB/TRA2B/PPIE/PLRG1/RBM8A/PRPF3/DHX38/LSM7/PPIH/PRPF6/PRPF31/SF3A2/CRNKL1/SF3B4/CHERP/SRSF1/THOC1/EIF4A3/DHX15/SNRNP200/DHX8/LSM4/PRPF19/SRSF10/HNRNPU/SNRPD1/XAB2
##                  Count
## REACTOME_2467813    45
## REACTOME_72163      48
## GO:0006364          36
## REACTOME_2500257    30
## KEGG_hsa03013       29
## KEGG_hsa03040       36
# gseRes2 = enrich.GSE(genelist)
# head(gseRes2@result)

2.4 Visualize enrichment results.

2.4.1 Barplot

require(ggplot2)
df = hgtRes1@result
df$logFDR = -log10(df$p.adjust)
p = BarView(df[1:5,], "Description", 'logFDR')
p = p + labs(x = NULL) + coord_flip()
p

# Or use function barplot from enrichplot package
barplot(hgtRes1, showCategory = 5)

2.4.2 Dot plot

## top: up-regulated pathways; 
## bottom: down-regulated pathways
EnrichedView(hgtRes1, top = 0, bottom = 5, mode = 1)

EnrichedView(hgtRes1, top = 0, bottom = 5, mode = 2)