Basic checks of BioPlex PPI data
3 March 2022
Package
BioPlex 1.0.2
1 Setup
library(BioPlex)
library(AnnotationDbi)
library(AnnotationHub)
library(graph)Connect to AnnotationHub:
ah <- AnnotationHub::AnnotationHub()Connect to ExperimentHub:
eh <- ExperimentHub::ExperimentHub()OrgDb package for human:
orgdb <- AnnotationHub::query(ah, c("orgDb", "Homo sapiens"))
orgdb <- orgdb[[1]]
orgdb
#> OrgDb object:
#> | DBSCHEMAVERSION: 2.1
#> | Db type: OrgDb
#> | Supporting package: AnnotationDbi
#> | DBSCHEMA: HUMAN_DB
#> | ORGANISM: Homo sapiens
#> | SPECIES: Human
#> | EGSOURCEDATE: 2021-Sep13
#> | EGSOURCENAME: Entrez Gene
#> | EGSOURCEURL: ftp://ftp.ncbi.nlm.nih.gov/gene/DATA
#> | CENTRALID: EG
#> | TAXID: 9606
#> | GOSOURCENAME: Gene Ontology
#> | GOSOURCEURL: http://current.geneontology.org/ontology/go-basic.obo
#> | GOSOURCEDATE: 2021-09-01
#> | GOEGSOURCEDATE: 2021-Sep13
#> | GOEGSOURCENAME: Entrez Gene
#> | GOEGSOURCEURL: ftp://ftp.ncbi.nlm.nih.gov/gene/DATA
#> | KEGGSOURCENAME: KEGG GENOME
#> | KEGGSOURCEURL: ftp://ftp.genome.jp/pub/kegg/genomes
#> | KEGGSOURCEDATE: 2011-Mar15
#> | GPSOURCENAME: UCSC Genome Bioinformatics (Homo sapiens)
#> | GPSOURCEURL:
#> | GPSOURCEDATE: 2021-Jul20
#> | ENSOURCEDATE: 2021-Apr13
#> | ENSOURCENAME: Ensembl
#> | ENSOURCEURL: ftp://ftp.ensembl.org/pub/current_fasta
#> | UPSOURCENAME: Uniprot
#> | UPSOURCEURL: http://www.UniProt.org/
#> | UPSOURCEDATE: Wed Sep 15 18:21:59 2021
keytypes(orgdb)
#> [1] "ACCNUM" "ALIAS" "ENSEMBL" "ENSEMBLPROT" "ENSEMBLTRANS"
#> [6] "ENTREZID" "ENZYME" "EVIDENCE" "EVIDENCEALL" "GENENAME"
#> [11] "GENETYPE" "GO" "GOALL" "IPI" "MAP"
#> [16] "OMIM" "ONTOLOGY" "ONTOLOGYALL" "PATH" "PFAM"
#> [21] "PMID" "PROSITE" "REFSEQ" "SYMBOL" "UCSCKG"
#> [26] "UNIPROT"2 Check: identify CORUM complexes that have a subunit of interest
Get core set of complexes:
core <- getCorum(set = "core", organism = "Human")Turn the CORUM complexes into a list of graph instances, where all nodes of a complex are connected to all other nodes of that complex with undirected edges.
core.glist <- corum2graphlist(core, subunit.id.type = "UNIPROT")Identify complexes that have a subunit of interest:
has.cdk2 <- hasSubunit(core.glist,
subunit = "CDK2",
id.type = "SYMBOL")Check the answer:
table(has.cdk2)
#> has.cdk2
#> FALSE TRUE
#> 2408 9
cdk2.glist <- core.glist[has.cdk2]
lapply(cdk2.glist, function(g) unlist(graph::nodeData(g, attr = "SYMBOL")))
#> $CORUM311_Cell_cycle_kinase_complex_CDK2
#> P12004 P24385 P24941 P38936
#> "PCNA" "CCND1" "CDK2" "CDKN1A"
#>
#> $`CORUM1003_RC_complex_(Replication_competent_complex)`
#> P09884 P20248 P24941 P35249 P35250 P35251 P40937 P40938 Q14181
#> "POLA1" "CCNA2" "CDK2" "RFC4" "RFC2" "RFC1" "RFC5" "RFC3" "POLA2"
#>
#> $`CORUM1004_RC_complex_during_S-phase_of_cell_cycle`
#> P09874 P09884 P11387 P15927 P18858 P20248 P24941 P27694 P28340 P35244
#> "PARP1" "POLA1" "TOP1" "RPA2" "LIG1" "CCNA2" "CDK2" "RPA1" "POLD1" "RPA3"
#> P35250 P35251 Q07864
#> "RFC2" "RFC1" "POLE"
#>
#> $`CORUM1656_p27-cyclinE-CDK2_complex`
#> P24864 P24941 P46527
#> "CCNE1" "CDK2" "CDKN1B"
#>
#> $`CORUM3015_p27-cyclinE-Cdk2_-_Ubiquitin_E3_ligase_(SKP1A,_SKP2,_CUL1,_CKS1B,_RBX1)_complex`
#> P24864 P24941 P46527 P61024 P62877 P63208 Q13309 Q13616
#> "CCNE1" "CDK2" "CDKN1B" "CKS1B" "RBX1" "SKP1" "SKP2" "CUL1"
#>
#> $`CORUM5556_CDK2-CCNA2_complex`
#> P20248 P24941
#> "CCNA2" "CDK2"
#>
#> $`CORUM5559_CDC2-CCNA2-CDK2_complex`
#> P06493 P20248 P24941
#> "CDK1" "CCNA2" "CDK2"
#>
#> $`CORUM5560_CDK2-CCNE1_complex`
#> P24864 P24941
#> "CCNE1" "CDK2"
#>
#> $`CORUM6589_E2F-1-DP-1-cyclinA-CDK2_complex`
#> P24941 P78396 Q01094 Q14186
#> "CDK2" "CCNA1" "E2F1" "TFDP1"We can then also inspect the graph with plotting utilities from the Rgraphviz package:
plot(cdk2.glist[[1]], main = names(cdk2.glist)[1])3 Check: extract BioPlex PPIs for a CORUM complex
Get the latest version of the 293T PPI network:
bp.293t <- getBioPlex(cell.line = "293T", version = "3.0")Turn the BioPlex PPI network into one big graph where bait and prey relationship are represented by directed edges from bait to prey.
bp.gr <- bioplex2graph(bp.293t)Now we can also easily pull out a BioPlex subnetwork for a CORUM complex of interest:
n <- graph::nodes(cdk2.glist[[1]])
bp.sgr <- graph::subGraph(n, bp.gr)
bp.sgr
#> A graphNEL graph with directed edges
#> Number of Nodes = 4
#> Number of Edges = 54 Check: identify interacting domains for a PFAM domain of interest
Add PFAM domain annotations to the node metadata:
bp.gr <- BioPlex::annotatePFAM(bp.gr, orgdb)Create a map from PFAM to UNIPROT:
unip2pfam <- graph::nodeData(bp.gr, graph::nodes(bp.gr), "PFAM")
pfam2unip <- stack(unip2pfam)
pfam2unip <- split(as.character(pfam2unip$ind), pfam2unip$values)
head(pfam2unip, 2)
#> $PF00001
#> [1] "P28566" "P25106" "P23945" "Q9HBX9" "P16473" "P04201" "Q9HC97" "P30968"
#> [9] "Q9Y2T6" "Q14330" "P46089" "Q15391" "Q9BXA5" "Q13304" "P61073" "P21462"
#> [17] "P25090" "Q99679" "P21730" "P30556" "P43088" "P32246" "P32249" "Q9Y2T5"
#> [25] "Q7Z602" "P43657" "O00398" "Q9H244" "Q86VZ1" "Q9NPB9" "Q99788" "P51684"
#> [33] "P35414" "O00590" "Q9H1Y3" "P55085" "O15218" "Q9GZQ4" "P25101" "Q9NS66"
#> [41] "Q9NQS5" "P21453" "P14416" "P24530" "P32239" "Q16581" "O00421" "Q9UHM6"
#> [49] "Q8N6U8" "P20309" "O15354" "Q9BXC0" "P47775" "P30550" "P49146" "P47900"
#> [57] "Q8TDU9" "P25103" "P35372" "P41597" "Q9P296" "P28335" "O95136" "P08173"
#> [65] "P29371" "P41146" "P43119" "O95977" "Q9HBW0" "Q99677" "Q9BXB1" "Q8WXD0"
#> [73] "O43193" "P30989" "Q8NGU9" "P47901" "P22888" "Q9GZN0" "P21917" "O60755"
#> [81] "Q8TDV0" "O43614" "Q9NS67" "P08912" "Q9UPC5" "Q8TDV2" "Q92633" "Q9NQ55"
#> [89] "Q13585" "Q9UBY5" "Q9H228" "P28222"
#>
#> $PF00002
#> [1] "Q8IZP9" "P41587" "Q8IZF4" "P49190" "P32241" "P47871" "P48960" "Q8IZF5"
#> [9] "O14514" "Q03431" "Q9NYQ6" "Q9HCU4" "Q8WXG9" "Q9NYQ7" "O60242" "O60241"
#> [17] "Q9HAR2" "O94910" "Q8IWK6" "O95490" "Q96PE1" "Q86SQ4"Let’s focus on PF02023, corresponding to the zinc finger-associated SCAN domain. For each protein containing the SCAN domain, we now extract PFAM domains connected to the SCAN domain by an edge in the BioPlex network.
scan.unip <- pfam2unip[["PF02023"]]
getIAPfams <- function(n) graph::nodeData(bp.gr, graph::edges(bp.gr)[[n]], "PFAM")
unip2iapfams <- lapply(scan.unip, getIAPfams)
unip2iapfams <- lapply(unip2iapfams, unlist)
names(unip2iapfams) <- scan.unipLooking at the top 5 PFAM domains most frequently connected to the SCAN domain by an edge in the BioPlex network …
pfam2iapfams <- unlist(unip2iapfams)
sort(table(pfam2iapfams), decreasing = TRUE)[1:5]
#> pfam2iapfams
#> PF02023 PF00096 PF01352 PF06467 PF00249
#> 208 169 99 14 8… we find PF02023, the SCAN domain itself, and PF00096, a C2H2 type zinc finger domain. This finding is consistent with results reported in the BioPlex 3.0 publication.
See also the PFAM domain-domain association analysis vignette for a more comprehensive analysis of PFAM domain associations in the BioPlex network.
5 Check: expressed genes are showing up as prey (293T cells)
Get RNA-seq data for HEK293 cells from GEO: GSE122425
se <- getGSE122425()
se
#> class: SummarizedExperiment
#> dim: 57905 6
#> metadata(0):
#> assays(2): raw rpkm
#> rownames(57905): ENSG00000223972 ENSG00000227232 ... ENSG00000231514
#> ENSG00000235857
#> rowData names(4): SYMBOL KO GO length
#> colnames(6): GSM3466389 GSM3466390 ... GSM3466393 GSM3466394
#> colData names(41): title geo_accession ... passages.ch1 strain.ch1Inspect expression of prey genes:
bait <- unique(bp.293t$SymbolA)
length(bait)
#> [1] 8995
prey <- unique(bp.293t$SymbolB)
length(prey)
#> [1] 10419ind <- match(prey, rowData(se)$SYMBOL)
par(las = 2)
boxplot(log2(assay(se, "rpkm") + 0.5)[ind,],
names = se$title,
ylab = "log2 RPKM")
How many prey genes are expressed (raw read count > 0) in all 3 WT reps:
# background: how many genes in total are expressed in all three WT reps
gr0 <- rowSums(assay(se)[,1:3] > 0)
table(gr0 == 3)
#>
#> FALSE TRUE
#> 33842 24063
# prey: expressed in all three WT reps
table(gr0[ind] == 3)
#>
#> FALSE TRUE
#> 599 9346
# prey: expressed in at least one WT rep
table(gr0[ind] > 0)
#>
#> FALSE TRUE
#> 305 9640Are prey genes overrepresented in the expressed genes?
exprTable <-
matrix(c(9346, 1076, 14717, 32766),
nrow = 2,
dimnames = list(c("Expressed", "Not.expressed"),
c("In.prey.set", "Not.in.prey.set")))
exprTable
#> In.prey.set Not.in.prey.set
#> Expressed 9346 14717
#> Not.expressed 1076 32766Test using hypergeometric test (i.e. one-sided Fisher’s exact test):
fisher.test(exprTable, alternative = "greater")
#>
#> Fisher's Exact Test for Count Data
#>
#> data: exprTable
#> p-value < 2.2e-16
#> alternative hypothesis: true odds ratio is greater than 1
#> 95 percent confidence interval:
#> 18.29105 Inf
#> sample estimates:
#> odds ratio
#> 19.34726Alternatively: permutation test, i.e. repeatedly sample number of prey genes from the background, and assess how often we have as many or more than 9346 genes expressed:
permgr0 <- function(gr0, nr.genes = length(prey))
{
ind <- sample(seq_along(gr0), nr.genes)
sum(gr0[ind] == 3)
}perms <- replicate(permgr0(gr0), 1000)
summary(perms)
#> Min. 1st Qu. Median Mean 3rd Qu. Max.
#> 1000 1000 1000 1000 1000 1000
(sum(perms >= 9346) + 1) / 1001
#> [1] 0.0009990016 Check: is there a relationship between prey frequency and prey expression level?
Check which genes turn up most frequently as prey:
prey.freq <- sort(table(bp.293t$SymbolB), decreasing = TRUE)
preys <- names(prey.freq)
prey.freq <- as.vector(prey.freq)
names(prey.freq) <- preys
head(prey.freq)
#> HSPA5 HSPA8 TUBB8 UBB YBX1 YWHAH
#> 199 192 176 173 139 132
summary(prey.freq)
#> Min. 1st Qu. Median Mean 3rd Qu. Max.
#> 1.00 2.00 6.00 11.34 16.00 199.00
hist(prey.freq, breaks = 50, main = "", xlab = "Number of PPIs", ylab = "Number of genes")
Prey genes are involved 11 PPIs on average.
There doesn’t seem to be a strong correlation between expression level and the frequency of gene to turn up as prey:
ind <- match(names(prey.freq), rowData(se)$SYMBOL)
rmeans <- rowMeans(assay(se, "rpkm")[ind, 1:3])
log.rmeans <- log2(rmeans + 0.5)
par(pch = 20)
plot( x = prey.freq,
y = log.rmeans,
xlab = "prey frequency",
ylab = "log2 RPKM")
cor(prey.freq,
log.rmeans,
use = "pairwise.complete.obs")
#> [1] 0.2035977See also the BioNet maximum scoring subnetwork analysis vignette for a more comprehensive analysis of the 293T transcriptome data from GSE122425 when mapped onto BioPlex PPI network.
7 Check: differential protein expression (HEK293 vs. HCT116)
Get the relative protein expression data comparing 293T and HCT116 cells from Supplementary Table S4A of the BioPlex 3 paper:
bp.prot <- getBioplexProteome()
bp.prot
#> class: SummarizedExperiment
#> dim: 9604 10
#> metadata(0):
#> assays(1): exprs
#> rownames(9604): P0CG40 Q8IXZ3-4 ... Q9H3S5 Q8WYQ3
#> rowData names(5): ENTREZID SYMBOL nr.peptides log2ratio adj.pvalue
#> colnames(10): HCT1 HCT2 ... HEK4 HEK5
#> colData names(1): cell.line
rowData(bp.prot)
#> DataFrame with 9604 rows and 5 columns
#> ENTREZID SYMBOL nr.peptides log2ratio adj.pvalue
#> <character> <character> <integer> <numeric> <numeric>
#> P0CG40 100131390 SP9 1 -2.819071 6.66209e-08
#> Q8IXZ3-4 221833 SP8 3 -3.419888 6.94973e-07
#> P55011 6558 SLC12A2 4 0.612380 4.85602e-06
#> O60341 23028 KDM1A 7 -0.319695 5.08667e-04
#> O14654 8471 IRS4 4 -5.951096 1.45902e-06
#> ... ... ... ... ... ...
#> Q9H6X4 80194 TMEM134 2 -0.379342 7.67195e-05
#> Q9BS91 55032 SLC35A5 1 -2.237634 8.75523e-05
#> Q9UKJ5 26511 CHIC2 1 -0.614932 1.78756e-03
#> Q9H3S5 93183 PIGM 1 -1.011397 8.91589e-06
#> Q8WYQ3 400916 CHCHD10 1 0.743852 1.17163e-03A couple of quick sanity checks:
- The relative abundances are scaled to sum up to 100% for each protein:
rowSums(assay(bp.prot)[1:5,])
#> P0CG40 Q8IXZ3-4 P55011 O60341 O14654
#> 99.99994 99.99991 99.99996 100.00011 100.00006- The
rowDatacolumnlog2ratiocorresponds to the mean of the five HEK samples, divided by the mean of the five HCT samples (and then taking log2 of it):
ratio <- rowMeans(assay(bp.prot)[1:5, 1:5]) / rowMeans(assay(bp.prot)[1:5, 6:10])
log2(ratio)
#> P0CG40 Q8IXZ3-4 P55011 O60341 O14654
#> -2.8190710 -3.4198879 0.6123799 -0.3196953 -5.9510960- The
rowDatacolumnadj.pvaluestores Benjamini-Hochberg adjusted p-values from a t-test between the five HEK samples and the five HCT samples:
t.test(assay(bp.prot)[1, 1:5], assay(bp.prot)[1, 6:10])
#>
#> Welch Two Sample t-test
#>
#> data: assay(bp.prot)[1, 1:5] and assay(bp.prot)[1, 6:10]
#> t = -27.898, df = 7.5779, p-value = 6.482e-09
#> alternative hypothesis: true difference in means is not equal to 0
#> 95 percent confidence interval:
#> -16.29035 -13.78047
#> sample estimates:
#> mean of x mean of y
#> 2.482288 17.517700The Transcriptome-Proteome analysis vignette also explores the agreement between differential gene expression and differential protein expression when comparing HEK293 against HCT116 cells.
8 SessionInfo
sessionInfo()
#> R version 4.1.2 (2021-11-01)
#> Platform: x86_64-w64-mingw32/x64 (64-bit)
#> Running under: Windows Server 2012 R2 x64 (build 9600)
#>
#> Matrix products: default
#>
#> locale:
#> [1] LC_COLLATE=C
#> [2] LC_CTYPE=English_United States.1252
#> [3] LC_MONETARY=English_United States.1252
#> [4] LC_NUMERIC=C
#> [5] LC_TIME=English_United States.1252
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] graph_1.72.0 AnnotationHub_3.2.2
#> [3] BiocFileCache_2.2.1 dbplyr_2.1.1
#> [5] AnnotationDbi_1.56.2 BioPlex_1.0.2
#> [7] SummarizedExperiment_1.24.0 Biobase_2.54.0
#> [9] GenomicRanges_1.46.1 GenomeInfoDb_1.30.1
#> [11] IRanges_2.28.0 S4Vectors_0.32.3
#> [13] BiocGenerics_0.40.0 MatrixGenerics_1.6.0
#> [15] matrixStats_0.61.0 BiocStyle_2.22.0
#>
#> loaded via a namespace (and not attached):
#> [1] bitops_1.0-7 bit64_4.0.5
#> [3] filelock_1.0.2 httr_1.4.2
#> [5] tools_4.1.2 bslib_0.3.1
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#> [29] htmltools_0.5.2 highr_0.9
#> [31] fastmap_1.1.0 limma_3.50.1
#> [33] rlang_1.0.1 RSQLite_2.2.10
#> [35] shiny_1.7.1 jquerylib_0.1.4
#> [37] generics_0.1.2 jsonlite_1.8.0
#> [39] dplyr_1.0.8 R.oo_1.24.0
#> [41] RCurl_1.98-1.6 magrittr_2.0.2
#> [43] GenomeInfoDbData_1.2.7 Matrix_1.4-0
#> [45] Rcpp_1.0.8 fansi_1.0.2
#> [47] lifecycle_1.0.1 R.methodsS3_1.8.1
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