SKA is a tool for comparing small and highly similar genomes using split k-mers. This guide will explain how to use SKA to build a phylogenetic tree for different Escherichia coli lineages in a few minutes. Although SKA is tailored more towards analysing variation within a lineage, tree-building ends up working fine for the whole species but requires more memory.

Why SKA is good for building phylogenetic trees

The basic approach to building a tree with SKA is to generate a SNP alignment using split k-mers and then feed that to a tree building algorithm of choice. Since SKA does not require a specifying a reference sequence to determine the SNPs, SKA gets around potential biases introduced by reference choice and is thus particularly well suited to analysing microbial genomes derived from outbreaks or pathogen surveillance.

Installing SKA

SKA can be installed in multiple ways:

1. Precompiled binary from the SKA GitHub repository.
2. Through the rust programming language’s package manager cargo with cargo install ska.
3. From bioconda with conda install -c bioconda ska2.
4. Building from source.

In this tutorial I used SKA v0.3.2 to run everything.

Input data

We will use a collection of 46 E. coli hybrid Nanopore+Illumina assemblies isolated from travellers visiting the city of Vientiane in Laos. These assemblies were published as part of Snaith et al. (2023).

All materials for running the analysis in this post can be downloaded from zenodo with wget

wget https://zenodo.org/record/8172518/files/building_trees_with_ska.tar

After downloading the assemblies, run tar -xvf building_trees_with_ska.tar to extract the files.

Reference-free workflow

The first example describes using a reference-free workflow to analyse assemblies in a case where we are only interested in the SNPs and do not care about structural rearrangements or other global changes. This is accomplished with the ska align command and the result can be used to e. g. build a tree for across-species variation.

Indexing the assemblies

To run the split k-mer alignment, we first need to build an index of the split k-mers from the assemblies. This is accomplished with the ska build command which requires a tab-separated list consisting of the sequence name and assembly file path as input:

LA002	assemblies/LA002.fa.gz
LA022	assemblies/LA022.fa.gz
LA023	assemblies/LA023.fa.gz
.
.
.

which is provided in data/laos_ska_input.tsv, or can be created using the command

paste <(ls assemblies | sed 's/[.].*$//g') <(ls -d assemblies/*) > data/laos_ska_input.tsv

The above only works if you’re using bash as your shell.

The split k-mers index is built from the input list by running

ska build -f data/laos_ska_input.tsv -k 31 -o output/laos_ska_index --threads 4

which will take a few minutes and use ~8GB of memory. SKA uses a lot of memory for species-wide analyses but when doing within-lineage analyses the method would be more efficient.

In the example we chose the value of k as 31 which is a commonly used choice for analysing bacterial genomes. For a more detailed analysis of the different possible values of k, have a look at the paper by Bussi, Kapon, and Reich in PLOS ONE.

Producing the SNP alignment

The SNP alignment can be extracted from the .skf file produced in the previous step by running

ska align --min-freq 1 --filter no-filter output/laos_ska_index.skf -o output/laos_ska_alignment.aln --threads 4

which will again take around a few minutes and use around 1.5GB memory. The resulting alignment has around 435k sites.

In the above command, the --min-freq option sets the maximum number of missing sites in the alignment to 1 and the --filter no-filter option specifies that no extra filtering should be performed. Other options include filtering all constant sites with --filter no-const or all constant and ambiguous sites with --filter no-ambig-or-const. Using the last option is equivalent to treating any ambiguous site as an N.

Build a tree

If no further pruning is needed, we can just use the alignment from the previous step to build a phylogeny. A good tool for doing this quickly is VeryFastTree but if you have a lot of time RAxML is also a good choice. Both can be installed from bioconda.

Build the tree with VeryFastTree (v4.0.1) by running

VeryFastTree -nt -gamma -gtr -threads 4 output/laos_ska_alignment.aln > output/laos_ska_tree.tree

As the name might suggest, VeryFastTree is fast and manages to build the tree in a few minutes.

Look at the tree

We can use the minimalistic plottree (install with pip install plottree) to quickly look at the tree using

plottree output/laos_ska_tree.tree

This shows that some of the assemblies are more closely related than the others.

Add in some colours

To build a fancier tree, we can use the R script provided at the end of this post to visualize and midpoint root the tree with the leaves coloured according to PopPUNK clusters provided in data/laos_poppunk_clusters.tsv. Adding the cluster colours and labels shows that SKA + VeryFastTree has managed to infer a phylogeny that preserves their locality (defined as two assemblies belonging to the same cluster).

Next, we’ll take a look at using the other mode SKA provides for producing alignments: mapping against a reference sequence.

Alignment-based workflow

In addition to the reference-free alignments, SKA can also be used to produce alignments against a reference genome. This is useful if the order of the output alignment is meaningful, such as when planning to use gubbins to remove recombination.

Note that gubbins is not suitable for analysing variation across a species (see the note about “What type of dataset can be analysed with Gubbins?” in the gubbins documentation for more details). To avoid running gubbins on unsuitable data, we’ll focus our analysis on variation within the largest PopPUNK cluster in the collection, SC4. This cluster corresponds to the E. coli ST10 clonal complex.

Reference sequence for SC4

Fortunately for us, ST10 has a downloadable a reference genome in the ENA that we can download with wget

wget -q -O - https://www.ebi.ac.uk/ena/browser/api/fasta/U00096.3?download=true | gzip > GCA_000005845.2.fna.gz

Since we now only want to use a subset of the assemblies, we’ll need to rebuild the index for the genomes that belong to SC4. This info is contained in the data/laos_poppunk_clusters.tsv file and we use the following commands to both convert it to an input list for SKA and to add the reference sequence to the index

paste <(grep "SC4$" data/laos_poppunk_clusters.tsv | cut -f1) <(grep "SC4$" data/laos_poppunk_clusters.tsv | cut -f1 | sed 's/^/assemblies\//g' | sed 's/$/.fa.gz/g') > output/laos_SC4_input_list.tsv
echo -e "GCA_000005845.2\t$(pwd)/GCA_000005845.2.fna.gz" >> output/laos_SC4_input_list.tsv

Aligning with ska map

Build the index as previously with ska build and then run ska map to align the indexed assemblies against the reference genome with

ska build -f output/laos_SC4_input_list.tsv -k 31 -o output/laos_SC4_index --threads 4
ska map -o output/laos_SC4_map.aln --ambig-mask --threads 4 GCA_000005845.2.fna.gz output/laos_SC4_index.skf

This time the alignment has 4 641 653 sites, which is the number of bases in the reference genome. Compared to ska align, using ska map keeps all bases in the reference sequence and replaces the sites it cannot find in the indexed assemblies with gaps. If you use the command

sed -n '4p' output/laos_SC4_map.aln | less

to look at the alignment of the first assembly against the reference you’ll see that some sites are marked as missing/gaps.

Removing recombination

In a typical genomic epidemiology workflow looking at variants within a lineage one typically wants to remove recombination from the alignment. For our SC4 alignment, we will do this using gubbins. Since Gubbins only accepts characters in the ACGTN- range, we had to use the --ambig-mask option with ska map to mask ambiguous bases (SKA uses the IUPAC notation which gubbins does not support) with N’s.

Gubbins (v3.2.1 in our example) can be run on the reference mapping with

run_gubbins.py --prefix output/gubbins output/laos_SC4_map.aln --threads 4 --filter-percentage 27.0

This will create the recombination-free tree output/gubbins.final_tree.tre. We had to increase the --filter-percentage parameter from the default 25.0 to 27.0 because one of the input sequences had ~26% missing sites.

Gubbins produces an alignment where recombination has been removed and only ~7 000 sites remain. These sites are enough to differentiate the assemblies belonging to SC4 and we can check this by visualising the tree with plottree

plottree output/gubbins.final_tree.tre

In this tree the assemblies from Laos cluster into several distinct groups within the lineage, and none of them are closely related to the reference genome which is sensible since the reference genome is from the E. coli K-12 strain that was isolated a hundred years ago on the other side of the world in Palo Alto, California, USA in 1922.

And that’s it! Happy tree-building.

About the author

I visited John Lees’ lab in Spring 2023 and have been using both SKA v1 and v2 to build lots of trees. This guide was a result of wanting to get others to do the same because installing snippy gets increasingly more difficult over time and reference-free approaches should be preferred anyway. If you want to know what I’m doing now, check out my personal website https://maklin.fi.

R script for plotting trees

#!/usr/bin/env Rscript
#
## Plot a tree and add some colors

library("ape")
library("phytools")

## Okabe-Ito color palette for the 7 most common clusters
colors <- c("#e69f00", "#56b4e9", "#009e73", "#f0e442", "#0072b2", "#d55e00", "#cc79a7")

## Read the raw SKA alignment + VeryFastTree tree in and midpoint root it
tree2 <- read.tree("ska.aln.tree")
midpoint_rooted_tree <- midpoint.root(tree2)

## Read in the PopPUNK clusters for each assembly
sts <- read.table("data/laos_poppunk_clusters.tsv", sep='\t', comment.char='@', header=FALSE)
sts <- sts[match(tree2$tip.label, sts$V1), ]

## Get the 7 most common clusters
top_st_names <- names(tail(sort(table(sts$V2)), 7))
top_st_colors <- cbind(top_st_names, colors[as.numeric(factor(top_st_names))])

## Set up the colors so that the 7 most common have a different color from the rest
other_st_names <- setdiff(unique(sts$V2), top_st_names)
st_colors <- rbind(top_st_colors, cbind(other_st_names, rep("black", length(other_st_names))))
st_colors <- st_colors[match(sts[, 2], st_colors), ]

## Add colors and labels, same as above
st_colors <- st_colors[match(sts[, 2], st_colors), ]
tree2$tip.label <- paste(tree2$tip.label, sts[, 2], sep='_')
tree2.colors <- st_colors[, 2]

## Plot both trees side-by-side
pdf(file="SKA_tree_fancy.pdf", width=9, height=9)
par(mar=c(1, 1, 3, 1))
plot.phylo(tree2, tip.color=tree2.colors)
title(main="Maximum likelihood tree\n(SKA + VeryFastTree)", adj=0, font.main=1, cex.main=1.5)
legend("bottomright", legend=top_st_colors[order(as.numeric(gsub("SC", "", top_st_colors[, 1]))), 1], fill=top_st_colors[order(as.numeric(gsub("SC", "", top_st_colors[, 1]))), 2], bty='n', title="PopPUNK\ncluster", cex=1.33)
dev.off()