Contents

1 Overview

Geneplast is designed for large-scale evolutionary analysis of orthologous groups, assessing the distribution of orthologous genes in a given species tree. Figure 1 illustrates the distribution of two hypothetical orthologous groups in 13 species. Diversity (Hα) and abundance (Dα) are useful metrics to describe the distribution of orthologous genes in a species tree. Geneplast calculates Hα assessing the normalized Shannon’s diversity (Castro et al. 2008). High Hα represents an homogeneous distribution (Figure 1a), while low Hα indicates that few species concentrate most of the observed orthologous genes (Figure 1b). The abundance Dα is given by the number of orthologous genes divided by the number of species in the tree. Geneplast uses Hα and Dα to calculate an evolutionary plasticity index (EPI) as defined by Dalmolin et al. (2011).

title Figure 1. Toy examples illustrating the distribution of orthologous genes in a given species tree. (a) OG of low abundance (Dα) and high diversity (Hα). This hypothetical OG comprises orthologous genes observed in all species of the tree, without apparent deletions or duplications. (b) Example of an OG observed in many species, but not all. Numbers in parentheses represent the orthologous genes in each species.

In order to interrogate the evolutionary root of a given gene, Geneplast implements a new algorithm called Bridge, which assesses the probability that an ortholog is present in each Last Common Ancestor (LCA) of a species in a given species tree. The method is designed to deal with large-scale queries in order to interrogate, for example, all genes annotated in a network (please refer to Castro et al. (2008) for additional examples).

To illustrate the rooting inference consider the evolutionary scenarios presented in Figure 2 for the same hypothetical OGs from Figure 1. These OGs comprise a number of orthologous genes distributed among 13 species, and the pattern of presence or absence is indicated by green and grey colours, respectively. Observe that in Figure 2a at least one ortholog is present in all extant species. To explain this common genetic trait, a possible evolutionary scenario could assume that the ortholog was present in the LCA of all extant species and was genetically transmitted up to the descendants. For this scenario, the evolutionary root might be placed at the bottom of the species tree (i.e. node j). A similar interpretation could be done for OG in Figure 2b, but with the evolutionary root placed at node f. The Bridge algorithm infers the most consistent rooting scenario for the observed orthologs in a given species tree, computing a consistency score called Dscore and an associated empirical p-value. The Dscore is an estimate of the stability of the inferred roots and the empirical p-value is computed by permutation analysis.

title Figure 2. Evolutionary rooting scenarios for the same toy examples depicted in Figure 1. (a, b) Red circles indicate the evolutionary roots that best explain the observed orthologs in the species tree.

2 Quick start

We will use the gpdata.gs dataset available from the Geneplast package to demonstrate the analysis workflow. This dataset includes four objects containing orthology annotation derived from the STRING database, release 9.1. The gpdata.gs dataset is to be used for demonstration purposes only as it represents a subset of the STRING database. Geneplast can use other sources of orthology information, provided that the input data is set according to the gpdata.gs dataset.

library(geneplast)
data(gpdata.gs)

2.1 Evolutionary diversity and abundance

Next we will calculate diversity and abundance of orthologous groups using the gplast workflow. In the first step we will create an OGP object using the gplast.preprocess function, which will check the consistency of the input data. Then we will use the gplast function to perform diversity and abundance analysis, estimating the evolutionary plasticity index as defined by Dalmolin et al. (2011). This example will assess orthology annotation available from the gpdata.gs dataset for 121 eukaryotic species.

1 - Create an object of class OGP.

ogp <- gplast.preprocess(cogdata=cogdata, sspids=sspids, cogids=cogids, verbose=FALSE)

2 - Run the gplast function.

ogp <- gplast(ogp, verbose=FALSE)

3 - Get results.

res <- gplast.get(ogp, what="results")
head(res)
##         abundance diversity plasticity
## KOG0011    1.7328    0.9532     0.2759
## KOG0028    3.1466    0.9207     0.4809
## KOG0034    4.1121    0.9216     0.5455
## KOG0037    2.8252    0.9116     0.4577
## KOG0045    7.3534    0.8965     0.6694
## KOG0192   26.9286    0.8284     0.8404

2.2 Evolutionary rooting

The rooting analysis starts by running the groot.preprocess function, which will check the consistency of the input data. The user should provide 1) a cogdata object, 2) a phyloTree object, and 3) set a reference species for which the evolutionary root of its genes will be inferred. Next, the groot function will perform the rooting analysis. The results are then retrieved by groot.get function. The pipeline returns the inferred root of each OG evaluated by the Bridge algorithm, including the Dscore and associated empirical p-value. Additionally, the groot.plot function allows the visualization of the inferred roots (e.g. Figure 3) and the LCAs of the reference species (Figure 4).

1 - Create an object of class OGR.

ogr <- groot.preprocess(cogdata=cogdata, phyloTree=phyloTree, spid="9606", verbose=FALSE)

2 - Run the groot function.

set.seed(1)
ogr <- groot(ogr, nPermutations=100, verbose=FALSE)
# Note: nPermutations is set to 100 for demonstration purposes; please set nPermutations=1000

3 - Get results.

res <- groot.get(ogr, what="results")
head(res)
##           Root Dscore   Pvalue AdjPvalue
## NOG251516    3   0.67 1.53e-07  2.17e-05
## NOG80202     4   1.00 2.15e-10  3.06e-08
## NOG72146     6   0.82 9.44e-07  1.34e-04
## NOG44788     6   0.56 8.51e-05  1.21e-02
## NOG39906     7   1.00 6.55e-08  9.30e-06
## NOG45364     9   0.83 2.13e-05  3.03e-03

4 - Check the inferred root of a given OG.

groot.plot(ogr, whichOG="NOG40170")
## PDF file 'gproot_NOG40170_9606LCAs.pdf' has been generated!

5 - Visualize the LCAs of the reference species in the analysis.

groot.plot(ogr, plot.lcas = TRUE)
## PDF file 'gproot_9606LCAs.pdf' has been generated!

title Figure 3. Inferred evolutionary rooting scenario for NOG40170. Monophyletic groups are ordered to show all branches of the tree below the queried species in the analysis.

title Figure 4. LCAs of the reference species in the analysis.

3 Case studies

3.1 High-throughput rooting inference

This example shows how to assess all OGs annotated for H. sapiens in the geneplast.data.string.v91 package.

1 - Load orthogy data from the geneplast.data.string.v91 package.

# source("https://bioconductor.org/biocLite.R")
# biocLite("geneplast.data.string.v91")
library(geneplast.data.string.v91)
data(gpdata_string_v91)

2 - Create an object of class OGR for a reference spid.

ogr <- groot.preprocess(cogdata=cogdata, phyloTree=phyloTree, spid="9606")

3 - Run the groot function and infer the evolutionary roots. Note: this step may take a long processing time due to the large number of OGs in the input data (nPermutations argument is set to 100 for demonstration purpose only).

ogr <- groot(ogr, nPermutations=100, verbose=TRUE)

3.2 Map rooting annotation to PPI networks

This example aims to show the evolutionary roots of a protein-protein interaction (PPI) network. The next steps show how to map evolutionary rooting annotation from Geneplast to a graph model. Note: Gene annotation from the input PPI network must match the gene annotation available from the orthology data; in this example, ENTREZ IDs are used to match the datasets.

1 - Load a PPI network and all packages required in this case study. The ppi.gs object provides PPI information from apoptosis and genome-stability genes (Castro et al. 2008). The ppi.gs is an object of class igraph and it will be used to set graph attributes following igraph syntax rules. For detais on how to handle igraph objects please see igraph and RedeR documentation.

library(RedeR)
library(igraph)
library(RColorBrewer)
data(ppi.gs)

2 - Map rooting annotation to the igraph object.

g <- ogr2igraph(ogr, cogdata, ppi.gs, idkey = "ENTREZ")

3 - Set colors for rooting annotations.

pal <- brewer.pal(9, "RdYlBu")
color_col <- colorRampPalette(pal)(25)
g <- att.setv(g=g, from="Root", to="nodeColor", 
              cols=color_col, na.col="grey80", 
              breaks=seq(1,25))

4 - Adjust some igraph aesthetic attributes.

g <- att.setv(g = g, from = "SYMBOL", to = "nodeAlias")
E(g)$edgeColor <- "grey80"
V(g)$nodeLineColor <- "grey80"

5 - Send the igraph object to the RedeR application.

rdp <- RedPort()
calld(rdp)
resetd(rdp)
addGraph(rdp, g)
addLegend.color(rdp, colvec=g$legNodeColor$scale, 
                size=15, labvec=g$legNodeColor$legend, 
                title="Roots represented in Fig4")
relax(rdp)

6 - Group apoptosis and genome-stability genes into containers.

myTheme <- list(nestFontSize=25, zoom=80, isNest=TRUE, gscale=65, theme=2)
nestNodes(rdp, nodes=V(g)$name[V(g)$Apoptosis==1], 
          theme=c(myTheme, nestAlias="Apoptosis"))
nestNodes(rdp, nodes=V(g)$name[V(g)$GenomeStability==1], 
          theme=c(myTheme, nestAlias="Genome Stability"))
relax(rdp, p1=50, p2=50, p3=50, p4=50, p5= 50)

title Figure 5. Inferred evolutionary roots of a protein-protein interaction network.

3.3 Map rooting annotation to regulatory networks

This example aims to show the evolutionary roots of regulons (Fletcher et al. 2013). The next steps show how to map evolutionary rooting annotation from Geneplast to a graph model. Note: Gene annotation from the input regulatory network must match the gene annotation available from the orthology data; in this example, ENTREZ IDs are used to match the datasets.

1 - Load a regulatory network and all packages required in this case study. The rtni1st object provides regulons available from the Fletcher2013b data package computed from breast cancer data (Fletcher et al. 2013).

library(RTN)
library(Fletcher2013b)
library(RedeR)
library(igraph)
library(RColorBrewer)
data("rtni1st")

2 - Extract two regulons from the rtni1st object into an igraph object.

regs <- c("FOXM1","PTTG1")
g <- tni.graph(rtni1st, gtype = "rmap", regulatoryElements = regs)

3 - Map rooting annotation to the igraph object.

g <- ogr2igraph(ogr, cogdata, g, idkey = "ENTREZ")

4 - Set colors for rooting annotations.

pal <- brewer.pal(9, "RdYlBu")
color_col <- colorRampPalette(pal)(25)
g <- att.setv(g=g, from="Root", to="nodeColor", 
              cols=color_col, na.col = "grey80", 
              breaks = seq(1,25))

5 - Adjust some igraph aesthetic attributes.

idx <- V(g)$SYMBOL %in% regs
V(g)$nodeFontSize[idx] <- 30
V(g)$nodeFontSize[!idx] <- 1
E(g)$edgeColor <- "grey80"
V(g)$nodeLineColor <- "grey80"

6 - Send the igraph object to the RedeR application.

rdp <- RedPort()
calld(rdp)
resetd(rdp)
addGraph(rdp, g, layout=NULL)
addLegend.color(rdp, colvec=g$legNodeColor$scale, 
                size=15, labvec=g$legNodeColor$legend, 
                title="Roots represented in Fig4")
relax(rdp, 15, 100, 20, 50, 10, 100, 10, 2)

title Figure 6. Evolutionary roots of FOXM1 and PTTG1 regulons.

4 Runtime performance of the rooting pipeline

#--- Load ggplot
library(ggplot2)
library(ggthemes)
library(egg)
library(data.table)

#--- Load cogdata
data(gpdata.gs)

#--- Get "OGs" that include a ref. species (e.g. "9606")
cogids <- unique(cogdata$cog_id[cogdata$ssp_id=="9606"])
length(cogids)
# [1] 142

#--- Make a function to check runtime for different input sizes
check.rooting.runtime <- function(n){
  cogids.subset <- cogids[1:n]
  cogdata.subset <- cogdata[cogdata$cog_id%in%cogids.subset,]
  rt1 <- system.time(
    ogr <- groot.preprocess(cogdata=cogdata.subset, phyloTree=phyloTree,
                            spid="9606", verbose=FALSE)
  )["elapsed"]
  rt2 <- system.time(
    ogr <- groot(ogr, nPermutations=100, verbose=FALSE)
  )["elapsed"]
  rtime <- c(rt1,rt2)
  names(rtime) <- c("runtime.preprocess","runtime.groot")
  return(rtime)
}
# check.rooting.runtime(n=5)

#--- Run check.rooting.runtime() for different input sizes (x3 iterations)
input_size <- seq.int(10,length(cogids),10)
iterations <- 1:3
elapsed_lt <- lapply(iterations, function(i){
  print(paste0("Iteration ",i))
  it <- sapply(input_size, function(n){
    print(paste0("- size...",n))
    check.rooting.runtime(n)
  })
})

#--- Get 'preprocess' runtime
runtime.preprocess <- sapply(elapsed_lt, function(lt){
  lt["runtime.preprocess",]
})
runtime.preprocess <- data.frame(InputSize=input_size, runtime.preprocess)
runtime.preprocess <- melt(as.data.table(runtime.preprocess), "InputSize")
colnames(runtime.preprocess) <- c("Input.Size","Iteration","Elapsed.Time")

#--- Get 'groot' runtime
runtime.groot <- sapply(elapsed_lt, function(lt){
  lt["runtime.groot",]
})
runtime.groot <- data.frame(InputSize=input_size, runtime.groot)
runtime.groot <- melt(as.data.table(runtime.groot), "InputSize")
colnames(runtime.groot) <- c("Input.Size","Iteration","Elapsed.Time")

#--- Plot runtime results
cls <- c("#69b3a2",adjustcolor("#69b3a2", alpha=0.5))
gg1 <- ggplot(runtime.preprocess, aes(x=Input.Size, y=Elapsed.Time)) +
  geom_smooth(method=loess, se=TRUE) +
  geom_point(color=cls[1], fill=cls[2], size=3, shape=21) +
  scale_x_continuous(breaks=pretty(runtime.preprocess$Input.Size)) +
  scale_y_continuous(breaks=pretty(runtime.preprocess$Elapsed.Time)) +
  theme_pander() + labs(title="groot.preprocess()") +
  xlab("Input size (n)") + ylab("Elapsed time (s)") +
  theme(aspect.ratio=1, plot.title=element_text(size=12))
gg2 <- ggplot(runtime.groot, aes(x=Input.Size, y=Elapsed.Time)) +
  geom_smooth(method=loess, se=TRUE) +
  geom_point(color=cls[1], fill=cls[2], size=3, shape=21) +
  scale_x_continuous(breaks=pretty(runtime.groot$Input.Size)) +
  scale_y_continuous(breaks=pretty(runtime.groot$Elapsed.Time)) +
  theme_pander() + labs(title="groot()") +
  xlab("Input size (n)") + ylab("Elapsed time (s)") +
  theme(aspect.ratio=1, plot.title=element_text(size=12))
grid.arrange(gg1, gg2, nrow = 1)
# pdf(file = "rooting_runtime.pdf", width = 7, height = 3)
# grid.arrange(gg1, gg2, nrow = 1)
# dev.off()

title Figure 7. Runtime performance of the rooting pipeline. Each point indicate the elapsed time to evaluate the ‘check.rooting.runtime()’ function, which assesses the execution time of the ‘groot.preprocess()’ and ‘groot()’ functions separatelly.

5 Orthology data packages

The geneplast.data package provides supporting data via AnnotationHub for the Geneplast evolutionary analyses. The geneplast.data package contains pre-processed data from different OG databases for use in the Geneplast package. In the current version, geneplast.data provides orthology information from STRING (https://string-db.org/), OMA Browser (https://omabrowser.org/), and OrthoDB (https://www.orthodb.org/).

6 Session information

## R version 4.3.1 (2023-06-16)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.3 LTS
## 
## Matrix products: default
## BLAS:   /home/biocbuild/bbs-3.18-bioc/R/lib/libRblas.so 
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
## 
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##  [3] LC_TIME=en_GB              LC_COLLATE=C              
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
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## 
## time zone: America/New_York
## tzcode source: system (glibc)
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
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## other attached packages:
## [1] geneplast_1.28.0 BiocStyle_2.30.0
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## [19] BiocManager_1.30.22 compiler_4.3.1      igraph_1.5.1       
## [22] pkgconfig_2.0.3     Rcpp_1.0.11         lattice_0.22-5     
## [25] digest_0.6.33       R6_2.5.1            parallel_4.3.1     
## [28] magrittr_2.0.3      bslib_0.5.1         tools_4.3.1        
## [31] cachem_1.0.8

References

Castro, Mauro AA, Rodrigo JS Dalmolin, Jose CF Moreira, Jose CM Mombach, and Rita MC de Almeida. 2008. “Evolutionary Origins of Human Apoptosis and Genome-Stability Gene Networks.” Nucleic Acids Research 36 (19): 6269–83. https://doi.org/10.1093/nar/gkn636.

Dalmolin, Rodrigo JS, Mauro AA Castro, Jose Rybarczyk-Filho, Luis Souza, Rita MC de Almeida, and Jose CF Moreira. 2011. “Evolutionary Plasticity Determination by Orthologous Groups Distribution.” Biology Direct 6 (1): 22. https://doi.org/10.1186/1745-6150-6-22.

Fletcher, Michael, Mauro Castro, Suet-Feung Chin, Oscar Rueda, Xin Wang, Carlos Caldas, Bruce Ponder, Florian Markowetz, and Kerstin Meyer. 2013. “Master Regulators of FGFR2 Signalling and Breast Cancer Risk.” Nature Communications 4: 2464. https://doi.org/10.1038/ncomms3464.