# Chapter 11 Gallery of reproducible examples

## 11.1 Visualizing pairwise nucleotide sequence distance with phylogenetic tree

This example reproduces Fig. 1 of (Chen et al. 2017). It extracts accession numbers from tip labels of the HPV58 tree and calculates pairwise nucleotide sequence distances. The distance matrix is visualized as dot and line plots. This example demonstrates the abilities of adding multiple layers to a specific panel. As illustrated on Figure 11.1, the facet_plot function displays sequence distances as a dot plot and then adds a layer of line plot to the same panel, i.e. Sequence Distance. In addition, the tree in facet_plot can be fully annotated with multiple layers (clade labels, bootstrap support values, etc). The source code of this example was firstly published in Supplemental File of (Yu et al. 2018).

library(tibble)
library(tidyr)
library(Biostrings)
library(treeio)
library(ggplot2)
library(ggtree)

clade <- c(A3 = 92, A1 = 94, A2 = 108, B1 = 156, B2 = 159, C = 163, D1 = 173, D2 = 176)
cols <- c(A1 = "#EC762F", A2 = "#CA6629", A3 = "#894418", B1 = "#0923FA",
B2 = "#020D87", C = "#000000", D1 = "#9ACD32",D2 = "#08630A")

## visualize the tree with tip labels and tree scale
p <- ggtree(tree, aes(color = group), ladderize = FALSE) %>% rotate(rootnode(tree)) +
geom_tiplab(aes(label = paste0("italic('", label, "')")), parse = TRUE, size = 2.5) +
geom_treescale(x = 0, y = 1, width = 0.002) +
scale_color_manual(values = c(cols, "black"), na.value = "black", name = "Lineage",
breaks = c("A1", "A2", "A3", "B1", "B2", "C", "D1", "D2")) +
guides(color = guide_legend(override.aes = list(size = 5, shape = 15))) +
theme_tree2(legend.position = c(.1, .88))
## Optional
## add labels for monophyletic (A, C and D) and paraphyletic (B) groups
p <- p + geom_cladelabel(94, "italic(A1)", color = cols[["A1"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0, 0.5), parse = TRUE) +
geom_cladelabel(108, "italic(A2)", color = cols[["A2"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = 0.5, parse = TRUE) +
geom_cladelabel(131, "italic(A3)", color = cols[["A3"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0.5, 0), parse = TRUE) +
geom_cladelabel(92, "italic(A)", color = "darkgrey", offset = .00315, align = TRUE,
offset.text = 0.0002, barsize = 2, fontsize = 5, parse = TRUE) +
geom_cladelabel(156, "italic(B1)", color = cols[["B1"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0, 0.5), parse = TRUE) +
geom_cladelabel(159, "italic(B2)", color = cols[["B2"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0.5, 0), parse = TRUE) +
geom_strip(65, 71, "italic(B)", color = "darkgrey", offset = 0.00315, align = TRUE,
offset.text = 0.0002, barsize = 2, fontsize = 5, parse = TRUE) +
geom_cladelabel(163, "italic(C)", color = "darkgrey", offset = .0031, align = TRUE,
offset.text = 0.0002, barsize = 3.2, fontsize = 5, parse = TRUE) +
geom_cladelabel(173, "italic(D1)", color = cols[["D1"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0, 0.5), parse = TRUE) +
geom_cladelabel(176, "italic(D2)", color = cols[["D2"]], offset = .003, align = TRUE,
offset.text = -.001, barsize = 1.2, extend = c(0.5, 0), parse = TRUE) +
geom_cladelabel(172, "italic(D)", color = "darkgrey", offset = .00315, align = TRUE,
offset.text = 0.0002, barsize = 2, fontsize = 5, parse = TRUE)
## Optional
## display support values
p <- p + geom_nodelab(aes(subset = (node == 92), label = "*"),
color = "black", nudge_x = -.001, nudge_y = 1) +
geom_nodelab(aes(subset = (node == 155), label = "*"),
color = "black", nudge_x = -.0003, nudge_y = -1) +
geom_nodelab(aes(subset = (node == 158), label = "95/92/1.00"),
color = "black", nudge_x = -0.0001, nudge_y = -1, hjust = 1) +
geom_nodelab(aes(subset = (node == 162), label = "98/97/1.00"),
color = "black", nudge_x = -0.0001, nudge_y = -1, hjust = 1) +
geom_nodelab(aes(subset = (node == 172), label = "*"),
color = "black", nudge_x = -.0003, nudge_y = -1) 
## extract accession numbers from tip labels
tl <- treetip.label acc <- sub("\\w+\\|", "", tl) names(tl) <- acc ## read sequences from GenBank directly into R ## and convert the object to DNAStringSet tipseq <- ape::read.GenBank(acc) %>% as.character %>% lapply(., paste0, collapse = "") %>% unlist %>% DNAStringSet ## align the sequences using muscle tipseq_aln <- muscle::muscle(tipseq) tipseq_aln <- DNAStringSet(tipseq_aln) ## calculate pairwise hamming distances among sequences tipseq_dist <- stringDist(tipseq_aln, method = "hamming") ## calculate percentage of differences tipseq_d <- as.matrix(tipseq_dist) / width(tipseq_aln[1]) * 100 ## convert the matrix to tidy data frame for facet_plot dd <- as_tibble(tipseq_d) ddseq1 <- rownames(tipseq_d)
td <- gather(dd,seq2, dist, -seq1)
td$seq1 <- tl[td$seq1]
td$seq2 <- tl[td$seq2]

g <- p$data$group
names(g) <- p$data$label
td$clade <- g[td$seq2]

## visualize the sequence differences using dot plot and line plot
## and align the sequence difference plot to the tree using facet_plot
p2 <- facet_plot(p, panel = "Sequence Distance", data = td, geom_point,
mapping = aes(x = dist, color = clade, shape = clade), alpha = .6) %>%
facet_plot(panel = "Sequence Distance", data = td, geom = geom_path,
mapping=aes(x = dist, group = seq2, color = clade), alpha = .6) +
scale_shape_manual(values = 1:8, guide = FALSE)

print(p2)

## 11.2 Displaying different symbolic points for bootstrap values.

We can cut the bootstrap values into several intervals, e.g. to indicate whether the clade is high, moderate or low support. Then we can use these intervals as categorical variable to set different color or shape of symbolic points to indicate the bootstrap values belong to which category.

library(treeio)
library(ggplot2)
library(ggtree)

root <- rootnode(tree)
ggtree(tree, color="black", size=1.5, linetype=1,  right=TRUE) +
geom_tiplab(size=4.5, hjust = -0.060, fontface="bold") +  xlim(0, 0.09) +
geom_point2(aes(subset=!isTip & node != root,
fill=cut(support, c(0, 700, 900, 1000))),
shape=21, size=4) +
theme_tree(legend.position=c(0.2, 0.2)) +
scale_fill_manual(values=c("white", "grey", "black"), guide='legend',
name='Bootstrap Percentage(BP)',
breaks=c('(900,1e+03]', '(700,900]', '(0,700]'),
labels=expression(BP>=90,70 <= BP * " < 90", BP < 70))

## 11.3 Phylogenetic tree with genome locus structure

The geom_motif is defined in ggtree and it is a wrapper layer of gggenes::geom_gene_arrow. The geom_motif can automatically adjust genomic alignment by selective gene (via the on parameter) and can label genes via the label parameter. In the following example, we use example_genes dataset provided by gggenes. As the dataset only provide genomic coordinations of a set of genes, a phylogeny for the genomes need to be firstly constructed. We calculate jaccard similarity based on the ratio of overlapping genes among genomes and correspondingly determine genome distance. BioNJ algorithm was applied to construct the tree (Figure 11.3).

library(dplyr)
library(ggplot2)
library(gggenes)
library(ggtree)

get_genes <- function(data, genome) {
filter(data, molecule == genome) %>% pull(gene)
}

g <- unique(example_genes[,1])
n <- length(g)
d <- matrix(nrow = n, ncol = n)
rownames(d) <- colnames(d) <- g
genes <- lapply(g, get_genes, data = example_genes)

for (i in 1:n) {
for (j in 1:i) {
jaccard_sim <- length(intersect(genes[[i]], genes[[j]])) /
length(union(genes[[i]], genes[[j]]))
d[j, i] <- d[i, j] <- 1 - jaccard_sim
}
}

tree <- ape::bionj(d)

p <- ggtree(tree, branch.length='none') +
geom_tiplab() + xlim_tree(5.5) +
geom_facet(mapping = aes(xmin = start, xmax = end, fill = gene),
data = example_genes, geom = geom_motif, panel = 'Alignment',
on = 'genE', label = 'gene', align = 'left') +
scale_fill_brewer(palette = "Set3") +
scale_x_continuous(expand=c(0,0)) +
theme(strip.text=element_blank(),
panel.spacing=unit(0, 'cm'))

facet_widths(p, widths=c(1,2))

### References

Chen, Zigui, Wendy C. S. Ho, Siaw Shi Boon, Priscilla T. Y. Law, Martin C. W. Chan, Rob DeSalle, Robert D. Burk, and Paul K. S. Chan. 2017. “Ancient Evolution and Dispersion of Human Papillomavirus 58 Variants.” Journal of Virology 91 (21): e01285–17. https://doi.org/10.1128/JVI.01285-17.

Yu, Guangchuang, Tommy Tsan-Yuk Lam, Huachen Zhu, and Yi Guan. 2018. “Two Methods for Mapping and Visualizing Associated Data on Phylogeny Using Ggtree.” Molecular Biology and Evolution 35 (12): 3041–3. https://doi.org/10.1093/molbev/msy194.