MAGeCKFlute - Functional enrichment analysis in MAGeCKFlute
26 October 2021
Abstract
CRISPR (clustered regularly interspaced short palindrome repeats) coupled with nuclease Cas9 (CRISPR/Cas9) screens represent a promising technology to systematically evaluate gene functions. Data analysis for CRISPR/Cas9 screens is a critical process that includes identifying screen hits and exploring biological functions for these hits in downstream analysis. We have previously developed two algorithms, MAGeCK (Wei Li and Liu. 2014) and MAGeCK-VISPR (Wei Li and Liu. 2015), to analyze CRISPR/Cas9 screen data in various scenarios. These two algorithms allow users to perform quality control, read count generation and normalization, and calculate beta score to evaluate gene selection performance. In downstream analysis, the biological functional analysis is required for understanding biological functions of these identified genes with different screening purposes. Here, We developed MAGeCKFlute for supporting downstream analysis. MAGeCKFlute provides several strategies to remove potential biases within sgRNA-level read counts and gene-level beta scores. The downstream analysis with the package includes identifying essential, non-essential, and target-associated genes, and performing biological functional category analysis, pathway enrichment analysis and protein complex enrichment analysis of these genes. The package also visualizes genes in multiple ways to benefit users exploring screening data. Collectively, MAGeCKFlute enables accurate identification of essential, non-essential, and targeted genes, as well as their related biological functions. This vignette explains the use of the package and demonstrates typical workflows.Contents
- 1 Input data - weighted gene list
- 2 Enrichment analysis methods
- 3 Session info
- References
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 0.392682 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
## REACTOME_2467813 REACTOME_2467813
## REACTOME_72163 REACTOME_72163
## REACTOME_2500257 REACTOME_2500257
## GO:0006364 GO:0006364
## KEGG_hsa03013 KEGG_hsa03013
## REACTOME_6791226 REACTOME_6791226
## Description
## REACTOME_2467813 Separation of Sister Chromatids
## REACTOME_72163 mRNA Splicing - Major Pathway
## REACTOME_2500257 Resolution of Sister Chromatid Cohesion
## GO:0006364 rRNA processing
## KEGG_hsa03013 RNA transport
## REACTOME_6791226 Major pathway of rRNA processing in the nucleolus and cytosol
## NES pvalue p.adjust
## REACTOME_2467813 -2.303989 2.323769e-18 2.086280e-14
## REACTOME_72163 -2.254939 4.743669e-16 2.129433e-12
## REACTOME_2500257 -2.235105 1.063287e-12 3.182062e-09
## GO:0006364 -2.231951 2.072293e-12 4.253777e-09
## KEGG_hsa03013 -2.222448 2.369001e-12 4.253777e-09
## REACTOME_6791226 -2.215848 5.177676e-12 7.729604e-09
## 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
## 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
## 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
## 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
## REACTOME_6791226 23246/1453/51202/705/28987/55759/27042/55781/26354/88745/22984/51118/10171/79707/10799/65083/54555/56915/6209/23160/2091/4691/79897/10969/5394/6187/23481/23517/6202/92856/54512/9136/55127/81887
## 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
## 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
## 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
## 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
## REACTOME_6791226 BOP1/CSNK1D/DDX47/BYSL/NOB1/WDR12/UTP25/RIOK2/GNL3/RRP36/PDCD11/UTP11/RCL1/NOL9/RPP40/NOL6/DDX49/EXOSC5/RPS15/WDR43/FBL/NCL/RPP21/EBNA1BP2/EXOSC10/RPS2/PES1/MTREX/RPS8/IMP4/EXOSC4/RRP9/HEATR1/LAS1L
## Count
## REACTOME_2467813 45
## REACTOME_72163 48
## REACTOME_2500257 30
## GO:0006364 36
## KEGG_hsa03013 29
## REACTOME_6791226 34# 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)## Warning: `guides(<scale> = FALSE)` is deprecated. Please use `guides(<scale> =
## "none")` instead.
EnrichedView(hgtRes1, top = 0, bottom = 5, mode = 2)