`vignettes/vignette-05-tree-sequences.Rmd`

`vignette-05-tree-sequences.Rmd`

In this vignette, we will show how to specify sampling events to record individuals in the tree-sequence output file (a procedure which is called “remembering” of individuals in the SLiM context) and how to perform simple analyses using *slendr*’s interface to the *tskit* Python library. We will demonstrate these features on a simulation of Neanderthal introgression into anatomically modern humans. Specifically, we will show how to estimate the amount of Neanderthal ancestry using \(f\)-statistics calculated directly on the tree-sequence data structure generated by a *slendr* model, all entirely from R.

First, let’s set up a simple *non-spatial* model of Neanderthal introgression using *slendr.* This is essentially the same procedure which we have shown in another vignette introducing non-spatial *slendr* models. This is no different from a spatial model, except that we left out the `map`

argument in calling `population()`

.

```
library(slendr)
# create the ancestor of everyone and a chimpanzee outgroup
# (we set both N = 1 to reduce the computational time for this model)
anc <- population("ANC", time = 6.5e6, N = 1)
chimp <- population("CH", parent = anc, time = 6e6, N = 1)
# two populations of anatomically modern humans: Africans and Europeans
afr <- population("AFR", parent = anc, time = 600e3, N = 10000)
eur <- population("EUR", parent = afr, time = 70e3, N = 5000)
# Neanderthal population splitting at 600 ky ago from modern humans
# (becomes extinct by 40 ky ago)
nea <- population("NEA", parent = anc, time = 600e3, N = 1000, remove = 40e3)
# 3% Neanderthal introgression into Europeans between 55-50 ky ago
gf <- geneflow(from = nea, to = eur, rate = 0.03, start = 55000, end = 50000)
model <- compile(
populations = list(anc, chimp, nea, afr, eur), geneflow = gf,
generation_time = 30,
dir = file.path(tempdir(), "introgression"), overwrite = TRUE
)
```

Here’s our toy model visualized as a graph. Not particularly illuminating in this simple example, but it’s always worth keeping in mind that such graph is embedded within every *slendr* model and can be always invoked to make sure the model you’re setting up is correct:

`plot_graph(model) + ggplot2::theme(legend.position = "none")`

Now that we have defined a model, how do we sample data from it? Ideally, we would like to to schedule sampling events at a given time, sampling a defined number of individuals from a given population. This is why *slendr* provides a function `sampling()`

which serves to define such sampling schedule automatically and enforces that only populations which are already (i.e. after their appearance in the simulation) or still (before they are removed from the simulation) present will be sampled from.

In our example, we want to sample two Neanderthal individuals (the older one being the Altai Neanderthal, the younger one Vindija Neanderthal). These two genomes are what we need to estimate Neanderthal ancestry proportion using a so-called \(f_4\)-ratio statistic (more on that below):

```
#> # A tibble: 2 × 7
#> time pop n y_orig x_orig y x
#> <int> <chr> <int> <lgl> <lgl> <lgl> <lgl>
#> 1 40000 NEA 1 NA NA NA NA
#> 2 70000 NEA 1 NA NA NA NA
```

As you can see, the `sampling()`

function simply accepts the vector of times at which remembering should be schedule, and then a list of pairs `(<slendr population>, <number of individuals>)`

encoding from which populations should how many individuals be remembered at time points given in the `times`

vector.

Next, we want to sample some present-day individuals: an outgroup representing a chimpanzee, and a couple of Africans and Europeans:

```
present_samples <- sampling(model, times = 0, list(chimp, 1), list(afr, 5), list(eur, 10))
present_samples
```

```
#> # A tibble: 3 × 7
#> time pop n y_orig x_orig y x
#> <int> <chr> <int> <lgl> <lgl> <lgl> <lgl>
#> 1 0 CH 1 NA NA NA NA
#> 2 0 AFR 5 NA NA NA NA
#> 3 0 EUR 10 NA NA NA NA
```

As you can see above, the `sampling()`

function returns a plain old data frame with a very simple structure with three columns: time, population name, and the number of individuals. This means that you can define sampling events using whatever input data you might already have available (such as radiocarbon-dated ancient DNA samples from an Excel sheet from some publication). For instance, there has been a lot of interest to estimate the trajectory of Neanderthal ancestry in Europe over time using ancient DNA data from anatomically modern human individuals (also called early modern humans, EMH) across the last couple of tens of thousands of years. We can simulate something close to the available EMH ancient DNA data set over the last 50 thousand years by running doing this:

```
emh_samples <- sampling(model, times = runif(n = 40, min = 10000, max = 40000), list(eur, 1))
emh_samples
```

```
#> # A tibble: 40 × 7
#> time pop n y_orig x_orig y x
#> <int> <chr> <int> <lgl> <lgl> <lgl> <lgl>
#> 1 10319 EUR 1 NA NA NA NA
#> 2 11187 EUR 1 NA NA NA NA
#> 3 11395 EUR 1 NA NA NA NA
#> 4 11529 EUR 1 NA NA NA NA
#> 5 11927 EUR 1 NA NA NA NA
#> 6 12675 EUR 1 NA NA NA NA
#> 7 13689 EUR 1 NA NA NA NA
#> 8 13744 EUR 1 NA NA NA NA
#> 9 14774 EUR 1 NA NA NA NA
#> 10 16361 EUR 1 NA NA NA NA
#> # … with 30 more rows
```

This samples a single ancient European individuals at randomly chosen times between 40 and 10 ky ago.

One nice feature of the `sampling()`

function is that it only schedules sampling events for a population, if that population is present in the simulation at a given time. This makes it possible to simply take a wide time range for sampling, specify all populations and sizes of the samples, and let the function generate sampling events only for populations present at each time. If for some reason a stricter control over sampling is required, this behavior can be switched off by setting `strict = TRUE`

like this:

```
# this attempts to sample a Neanderthal individual at a point when Neanderthals
# are already extinct, resulting in an error
sampling(model, times = 10000, list(nea, 1), strict = TRUE)
```

`Error: Cannot schedule sampling for 'NEA' at time 10000 because the population will not be present in the simulation at that point. Consider running this function with `strict = FALSE` which will automatically retain only valid sampling events.`

Now that we already have the `model`

object ready, we can simulate data from it, sampling individuals according to our sampling schedule. We do this by calling the `slim()`

function as usual and specifying the sampling events with the `sampling =`

argument. Note that we bind the individual sampling schedule data frames using the `rbind`

function provided by base R (as we show above, the sampling schedule really is just a data frame and we can manipulate it as such):

```
slim(
model, sequence_length = 10e6, recombination_rate = 1e-8,
sampling = rbind(nea_samples, present_samples, emh_samples),
method = "batch", verbose = TRUE, seed = 314159
)
```

```
#> --------------------------------------------------
#> SLiM command to be executed:
#>
#> /Users/martin_petr/.my_local/bin/slim \
#> -d SEED=314159 \
#> -d 'SAMPLES="/var/folders/hr/_t1b0f5n7c76yrfsg8yk9l100000gn/T//RtmpX3ZX1w/filee2536f1b2b45"' \
#> -d 'MODEL="/var/folders/hr/_t1b0f5n7c76yrfsg8yk9l100000gn/T//RtmpX3ZX1w/introgression"' \
#> -d 'OUTPUT="/var/folders/hr/_t1b0f5n7c76yrfsg8yk9l100000gn/T//RtmpX3ZX1w/introgression/output"' \
#> -d SPATIAL=F \
#> -d SEQUENCE_LENGTH=10000000 \
#> -d RECOMB_RATE=1e-08 \
#> -d BURNIN_LENGTH=0 \
#> -d SIMULATION_LENGTH=216667 \
#> -d SAVE_LOCATIONS=F \
#> -d MAX_ATTEMPTS=1 \
#> /var/folders/hr/_t1b0f5n7c76yrfsg8yk9l100000gn/T//RtmpX3ZX1w/introgression/script.slim
#> --------------------------------------------------
```

When we execute the model, *slendr* will instruct SLiM to save the output of the simulation as a tree sequence file. By default, the file is stored in the model directory:

```
ts_file <- file.path(model$path, "output_ts.trees")
file.exists(ts_file)
```

`#> [1] TRUE`

Tree-sequences are one of the most revolutionary developments in population genetics in the last couple of decades for a number of reasons. One of them is the possibility to store extremely large data sets succinctly by encoding the entire evolutionary history of a sample of individuals as a series of correlated tree genealogies along the genome.

Going into too much detail on this topic is clearly beyond the scope of this tutorial, especially because everything is explain much better elsewhere. Instead, what we will demonstrate in the rest of this vignette is how you can access and manipulate tree-sequence outputs generated by *slendr* models and perform various statistics on them using Python modules *tskit* and *pyslim* directly from *slendr,* without having to leave R! The key is a magical R package *reticulate* which creates a seamless binding of Python modules with R. This means that even if you don’t know Python, *slendr* allows you to do do quite a lot with tree-sequences in R.

Of course, if you are a proficient Python user, it needs to be said that once you have the tree-sequence file generated by *slendr* & SLiM, you can easily perform every conceivable analysis directly using *tskit*. The intention here is to show how you can continue working on the tree-sequence files in R even after you have run the entire *slendr* simulation.

First, in order to be able to interface with *tskit* and *pyslim* using the *reticulate* package, you will need a working Python environment with the required Python modules `pyslim`

, `tskit`

and `msprime`

already installed. I personally manage Python installations and separate package environments using pyenv and pyenv-virtualenv. However, this is only my own personal preference. If you prefer using conda or any other Python management solution, feel free to use that. In any case, I recommend using tooling which is supported by reticulate itself, because it will be easy to find support should you run into any trouble.

For completeness, here is probably the simplest possible way to get started, assuming you have a reasonably up-to-date version of Python available at your system. We will use the built-in Python virtual environment management module `venv`

which is available at least since Python version 3.5, put it under the `~/.venvs/retipy`

so that it doesn’t clutter out home directory, and install the required Python modules into it:

```
# 1. create a separate virtual environment called 'retipy'
python3 -m venv retipy ~/.venvs/retipy
# 2. activate it for the current shell session
source ~/.venvs/retipy/bin/activate
# 3. install the required modules into this fresh environment
python3 -m pip install tskit pyslim msprime
```

That’s it! By creating a separate new environment and installing the modules into it, we’re making sure that we don’t cause problems for other Python projects or scripts you might already have and which might potentially use different versions of those modules.

Now we can instruct the *reticulate* package to use the newly created Python environment (if you use conda, pyenv-virtualenv or one of the other solutions for managing virtual environments, take a look here for a specific function to use in your case):

`reticulate::use_virtualenv("~/.venvs/retipy", required = TRUE)`

We can make sure that *reticulate* is indeed using our environment by calling:

`reticulate::py_config()`

```
#> python: /Users/martin_petr/.venvs/retipy/bin/python
#> libpython: /Library/Frameworks/Python.framework/Versions/3.9/lib/python3.9/config-3.9-darwin/libpython3.9.dylib
#> pythonhome: /Users/martin_petr/.venvs/retipy:/Users/martin_petr/.venvs/retipy
#> version: 3.9.0 (v3.9.0:9cf6752276, Oct 5 2020, 11:29:23) [Clang 6.0 (clang-600.0.57)]
#> numpy: /Users/martin_petr/.venvs/retipy/lib/python3.9/site-packages/numpy
#> numpy_version: 1.21.4
#> tskit: /Users/martin_petr/.venvs/retipy/lib/python3.9/site-packages/tskit
#>
#> NOTE: Python version was forced by use_python function
```

See this guide for more information about setting up R to work with different Python environment management tools, including conda.

With the technicalities out of the way, we can now load the tree-sequence file saved by SLiM using the *slendr* function `ts_load()`

. Optionally, we can also instruct this function to simplify the tree-sequence to only the individuals that we explicitly sampled (recall the sampling schedule we set up with the `sampling()`

function above). What *slendr* does here is provide an R-friendly interface to *tskit* and *pyslim*, essentially reproducing steps in this tutorial in a single R command. Note that we have to provide the `model`

object generated by `compile()`

above in order to have all model annotation information for the simulated tree-sequence data (we have to do this only once, and only during loading):

```
ts <- ts_load(model)
ts
```

```
#> ╔═══════════════════════════╗
#> ║TreeSequence ║
#> ╠═══════════════╤═══════════╣
#> ║Trees │ 56441║
#> ╟───────────────┼───────────╢
#> ║Sequence Length│ 10000000║
#> ╟───────────────┼───────────╢
#> ║Time Units │generations║
#> ╟───────────────┼───────────╢
#> ║Sample Nodes │ 30088║
#> ╟───────────────┼───────────╢
#> ║Total Size │ 20.4 MiB║
#> ╚═══════════════╧═══════════╝
#> ╔═══════════╤══════╤════════╤════════════╗
#> ║Table │Rows │Size │Has Metadata║
#> ╠═══════════╪══════╪════════╪════════════╣
#> ║Edges │268943│ 8.2 MiB│ No║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Individuals│ 71917│ 6.9 MiB│ Yes║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Migrations │ 0│ 8 Bytes│ No║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Mutations │ 0│ 1.2 KiB│ No║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Nodes │ 90201│ 3.3 MiB│ Yes║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Populations│ 5│ 2.6 KiB│ Yes║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Provenances│ 1│31.8 KiB│ No║
#> ╟───────────┼──────┼────────┼────────────╢
#> ║Sites │ 0│16 Bytes│ No║
#> ╚═══════════╧══════╧════════╧════════════╝
```

We can see that the tree-sequence data is very large and contains many more individuals than we need (much more than we explicitly specified for sampling): there are in total 71917 individuals in the tree-sequence which is a lot more than the 58 we scheduled for sampling! To avoid this issue, `ts_load()`

accepts an optional argument `simplify`

which triggers the simplification process to narrow down the set of recorded individuals just to those that we explicitly remembered:

`ts <- ts_load(model, simplify = TRUE)`

```
#> Warning: Simplifying a non-recapitated tree sequence. Make sure this is what you
#> really want
```

`ts`

```
#> ╔═══════════════════════════╗
#> ║TreeSequence ║
#> ╠═══════════════╤═══════════╣
#> ║Trees │ 17204║
#> ╟───────────────┼───────────╢
#> ║Sequence Length│ 10000000║
#> ╟───────────────┼───────────╢
#> ║Time Units │generations║
#> ╟───────────────┼───────────╢
#> ║Sample Nodes │ 116║
#> ╟───────────────┼───────────╢
#> ║Total Size │ 3.8 MiB║
#> ╚═══════════════╧═══════════╝
#> ╔═══════════╤═════╤═════════╤════════════╗
#> ║Table │Rows │Size │Has Metadata║
#> ╠═══════════╪═════╪═════════╪════════════╣
#> ║Edges │65125│ 2.0 MiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Individuals│ 9643│943.5 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Migrations │ 0│ 8 Bytes│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Mutations │ 0│ 1.2 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Nodes │ 9705│360.8 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Populations│ 5│ 2.6 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Provenances│ 2│ 32.3 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Sites │ 0│ 16 Bytes│ No║
#> ╚═══════════╧═════╧═════════╧════════════╝
```

By default, simplification in `ts_load()`

simplified the tree-sequence down to only remembered individuals (i.e. those we explicitly scheduled for sampling). We can also narrow down the simplification to a defined set of individuals using the `simplify_to =`

argument. Internally, simplification is implemented in a dedicated function `ts_simplify()`

which we can always call explicitly, like this:

`ts_small <- ts_simplify(ts, simplify_to = c("CH_1", "NEA_1", "NEA_2", "AFR_1", "AFR_2", "EUR_20", "EUR_50"))`

```
#> Warning: Simplifying a non-recapitated tree sequence. Make sure this is what you
#> really want
```

`ts_small`

```
#> ╔═══════════════════════════╗
#> ║TreeSequence ║
#> ╠═══════════════╤═══════════╣
#> ║Trees │ 7000║
#> ╟───────────────┼───────────╢
#> ║Sequence Length│ 10000000║
#> ╟───────────────┼───────────╢
#> ║Time Units │generations║
#> ╟───────────────┼───────────╢
#> ║Sample Nodes │ 14║
#> ╟───────────────┼───────────╢
#> ║Total Size │ 1.5 MiB║
#> ╚═══════════════╧═══════════╝
#> ╔═══════════╤═════╤═════════╤════════════╗
#> ║Table │Rows │Size │Has Metadata║
#> ╠═══════════╪═════╪═════════╪════════════╣
#> ║Edges │24698│771.8 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Individuals│ 3651│358.4 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Migrations │ 0│ 8 Bytes│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Mutations │ 0│ 1.2 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Nodes │ 3659│136.5 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Populations│ 5│ 2.6 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Provenances│ 3│ 32.8 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Sites │ 0│ 16 Bytes│ No║
#> ╚═══════════╧═════╧═════════╧════════════╝
```

Similarly, *slendr* provides a function `ts_recapitate()`

which performs recapitation. Again, we can do this in one go, by specifying `recapitate = TRUE`

in the call to `ts_load()`

after specifying a couple of additional arguments required for recapitation (see the pyslim documentation in the recapitation section for more detail):

```
ts <- ts_load(model, recapitate = TRUE, simplify = TRUE,
recombination_rate = 1e-8, Ne = 10000)
```

If we were to run this code chunk, we would get a message informing that the recapitation was not necessary because all genealogies in the tree-sequence are already coalesced (i.e. have each only one root node). We can do this check ourselves by calling another *slendr* function `ts_coalesced()`

which verifies that all trees in the sequence are fully coalesced:

`ts_coalesced(ts)`

`#> [1] TRUE`

You might have noticed that we did not simulate any mutations during the SLiM run. This is for computational efficiency. Luckily, the tree-sequence contains the complete history of a sample of individuals which makes it very easy to sprinkle mutations on the genealogies after this simulation is over. We can add mutations a given rate by running:

```
ts <- ts_mutate(ts, mutation_rate = 1e-8, random_seed = 314159)
ts
```

```
#> ╔═══════════════════════════╗
#> ║TreeSequence ║
#> ╠═══════════════╤═══════════╣
#> ║Trees │ 17204║
#> ╟───────────────┼───────────╢
#> ║Sequence Length│ 10000000║
#> ╟───────────────┼───────────╢
#> ║Time Units │generations║
#> ╟───────────────┼───────────╢
#> ║Sample Nodes │ 116║
#> ╟───────────────┼───────────╢
#> ║Total Size │ 7.2 MiB║
#> ╚═══════════════╧═══════════╝
#> ╔═══════════╤═════╤═════════╤════════════╗
#> ║Table │Rows │Size │Has Metadata║
#> ╠═══════════╪═════╪═════════╪════════════╣
#> ║Edges │65125│ 2.0 MiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Individuals│ 9643│943.5 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Migrations │ 0│ 8 Bytes│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Mutations │58284│ 2.1 MiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Nodes │ 9705│360.8 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Populations│ 5│ 2.6 KiB│ Yes║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Provenances│ 3│ 33.1 KiB│ No║
#> ╟───────────┼─────┼─────────┼────────────╢
#> ║Sites │58089│ 1.4 MiB│ No║
#> ╚═══════════╧═════╧═════════╧════════════╝
```

Even this step can be performed in one go throught the `ts_load`

function like this:

```
ts <- ts_load(model, recapitate = TRUE, simplify = TRUE, random_seed = 314159,
recombination_rate = 1e-8, Ne = 10000, mutation_rate = 1e-8)
```

Having done that, we can calculate some basic statistics on our simulated data.

However, before we do that, we would first like to note that everything that we do in the rest of this vignette (i.e. whenever we call a function with the prefix `ts_*()`

in *slendr*), we are interfacing with the *tskit* Python module under the hood. Our goal is to capture most of the analyses one might want to perform on tree-sequences in R and wrap them in a neat interface indistinguishable from any other R function—this is, after all, the reason why *reticulate* has been created in the first place (making various Python data science modules appear as if they were regular R packages).

**Note:** The visualisation of trees and tree-sequences in *slendr* is currently still work-in-progress. For a much more comprehensive overview of SVG plotting in *tskit* see the official tutorial on the subject.

We have spend a lot of time talking about how powerful the *tskit* library is. Another great example is tree(-sequence) visualisation. *tskit* implements visualisation of data in an SVG format which we can use for quick interactive plotting directly from the R console. First, we introduce a function `ts_tree()`

which allows us to extract one tree from the tree-sequence (either an *i*-th tree in the sequence, or a tree overlapping an *i*-th position of the simulated genome, depending on the value of its `mode`

argument):

```
# take the 1st tree in the sequence (mode = "index" by default)
tree <- ts_tree(ts_small, i = 1)
tree
```

```
#> ╔═════════════════════════════════╗
#> ║Tree ║
#> ╠═══════════════════╤═════════════╣
#> ║Index │ 0║
#> ╟───────────────────┼─────────────╢
#> ║Interval │0-2230 (2230)║
#> ╟───────────────────┼─────────────╢
#> ║Roots │ 1║
#> ╟───────────────────┼─────────────╢
#> ║Nodes │ 27║
#> ╟───────────────────┼─────────────╢
#> ║Sites │ 0║
#> ╟───────────────────┼─────────────╢
#> ║Mutations │ 0║
#> ╟───────────────────┼─────────────╢
#> ║Total Branch Length│ 472370║
#> ╚═══════════════════╧═════════════╝
```

We can then visualize the tree by calling:

`ts_draw(tree, width = 1000, height = 600)`

If we specify `labels = TRUE`

and provide the tree-sequence object itself (because that carries all the required model metadata such as individual names), we get a slightly more readable figure. We can also provide keyword arguments specific to *tskit* `draw_svg()`

function, such as `time_scale`

here:

`ts_draw(tree, width = 1000, height = 600, labels = TRUE, ts = ts_small, time_scale = "rank")`

As we mentioned in the previous section, the goal of this vignette is to show how to use *slendr* to perform the main tree-sequence operations using a convenient R interface to *tskit*. However, you should always keep in mind that you are not restricted to a subset of *tskit* functionality that *slendr* translated to R (i.e. all functions with the prefix `ts_`

). Thanks to the incredible R package *reticulate*, you can access Python methods and object variables directly, using the `$`

operator.

As an example, instead of calling the function `ts_coalesced()`

on the tree-sequence as we did above, we could check that all trees are coalesced by running the following snippet instead (note that this is very inefficient and we’re only doing the operation for the first one hundred trees):

```
# iterate over all trees in the tree-sequence and check if each
# has only one root (i.e. is fully coalesced) - note that Python
# lists are 0-based, which is something we need to take care of
all(sapply(seq_len(ts$num_trees)[1:100],
function(i) ts$at_index(i - 1)$num_roots == 1))
```

`#> [1] TRUE`

We believe it makes sense to use the R interface whenever possible (even if only because it makes many operations a little bit more convenient). However, if there is some functionality in *slendr* missing, you can always resort to accessing the Python objects directly as we have just demonstrated. You can verify that all methods and attributes of a Python tree-sequence object is still accessible in R:

`names(ts)`

```
#> [1] "alignments" "allele_frequency_spectrum"
#> [3] "as_fasta" "as_nexus"
#> [5] "aslist" "at"
#> [7] "at_index" "breakpoints"
#> [9] "coiterate" "count_topologies"
#> [11] "delete_intervals" "delete_sites"
#> [13] "diffs" "discrete_genome"
#> [15] "discrete_time" "divergence"
#> [17] "diversity" "draw_svg"
#> [19] "draw_text" "dump"
#> [21] "dump_tables" "dump_text"
#> [23] "edge" "edge_diffs"
#> [25] "edges" "edgesets"
#> [27] "equals" "f2"
#> [29] "f3" "f4"
#> [31] "file_uuid" "first"
#> [33] "first_generation_individuals" "Fst"
#> [35] "genealogical_nearest_neighbours" "general_stat"
#> [37] "genetic_relatedness" "genotype_matrix"
#> [39] "get_ll_tree_sequence" "get_num_mutations"
#> [41] "get_num_nodes" "get_num_records"
#> [43] "get_num_sites" "get_num_trees"
#> [45] "get_pairwise_diversity" "get_population"
#> [47] "get_sample_size" "get_samples"
#> [49] "get_sequence_length" "get_time"
#> [51] "haplotypes" "has_individual_parents"
#> [53] "has_reference_sequence" "ibd_segments"
#> [55] "individual" "individual_ages"
#> [57] "individual_ages_at" "individual_locations"
#> [59] "individual_parents" "individual_populations"
#> [61] "individual_times" "individuals"
#> [63] "individuals_alive_at" "kc_distance"
#> [65] "keep_intervals" "last"
#> [67] "ll_tree_sequence" "load"
#> [69] "load_tables" "ltrim"
#> [71] "max_root_time" "mean_descendants"
#> [73] "metadata" "metadata_schema"
#> [75] "migration" "migrations"
#> [77] "model_type" "mutation"
#> [79] "mutation_at" "mutations"
#> [81] "nbytes" "newick_trees"
#> [83] "node" "nodes"
#> [85] "nucleotide_at" "num_edges"
#> [87] "num_individuals" "num_migrations"
#> [89] "num_mutations" "num_nodes"
#> [91] "num_populations" "num_provenances"
#> [93] "num_samples" "num_sites"
#> [95] "num_trees" "pairwise_diversity"
#> [97] "parse_windows" "population"
#> [99] "populations" "provenance"
#> [101] "provenances" "recapitate"
#> [103] "records" "reference_sequence"
#> [105] "rtrim" "sample_count_stat"
#> [107] "sample_size" "samples"
#> [109] "segregating_sites" "sequence_length"
#> [111] "simplify" "site"
#> [113] "sites" "slim_generation"
#> [115] "slim_provenance" "slim_provenances"
#> [117] "slim_time" "subset"
#> [119] "table_metadata_schemas" "tables"
#> [121] "tables_dict" "Tajimas_D"
#> [123] "time_units" "to_macs"
#> [125] "to_nexus" "trait_correlation"
#> [127] "trait_covariance" "trait_linear_model"
#> [129] "trait_regression" "trees"
#> [131] "trim" "union"
#> [133] "variants" "write_fasta"
#> [135] "write_nexus" "write_vcf"
#> [137] "Y1" "Y2"
#> [139] "Y3"
```

In fact, you will recognize some of the elements in the output above from examples involving `ts_`

functions in this vignette! In short, there is no blackbox—*slendr* only provides a slightly more convenient layer over *tskit* for R users.

In addition to being a revolutionary breakthrough in terms of computation efficiency, many statistics that we are often interested in population genetics are a natural consequence of having a direct access to tree sequence genealogies, simply because those genealogies capture the true demographic history of a sample. Again, we can’t go into too much detail here but we encourage you to take a look at a paper by Ralph *et al.* on the duality between statistics expressed in terms of branch lengths and the traditional summaries based on samples of genetic variation.

For instance, we have functions such as `ts_f2()`

, `ts_f3()`

, `ts_f4()`

and `ts_f4ratio()`

which calculate the well-known set of Patterson’s \(f\)-statistics:

```
# f2 is a measure of the branch length connecting A and B
ts_f2(ts, A = "EUR_1", B = "AFR_1")
```

```
#> # A tibble: 1 × 3
#> A B f2
#> <chr> <chr> <dbl>
#> 1 EUR_1 AFR_1 0.0000213
```

```
# f4 is a measure of the drift shared between A and B after their split from C
ts_f3(ts, A = "EUR_1", B = "AFR_1", C = "CH_1")
```

```
#> # A tibble: 1 × 4
#> A B C f3
#> <chr> <chr> <chr> <dbl>
#> 1 EUR_1 AFR_1 CH_1 0.0000032
```

```
# this value should be very close to zero (no introgression in Africans)
ts_f4(ts, "AFR_1", "AFR_2", "NEA_1", "CH_1", mode = "branch")
```

```
#> # A tibble: 1 × 5
#> W X Y Z f4
#> <chr> <chr> <chr> <chr> <dbl>
#> 1 AFR_1 AFR_2 NEA_1 CH_1 0.0145
```

```
# this value should be significantly negative (many more ABBA sites
# compared to BABA site due to the introgression into Europeans)
ts_f4(ts, "AFR_1", "EUR_1", "NEA_1", "CH_1", mode = "branch")
```

```
#> # A tibble: 1 × 5
#> W X Y Z f4
#> <chr> <chr> <chr> <chr> <dbl>
#> 1 AFR_1 EUR_1 NEA_1 CH_1 -595.
```

These functions accept a `mode =`

argument, specifying whether the statistics should be calculated using mutation site patterns (`mode = "site"`

, the default), branch lengths (`mode = "branch"`

), or for each node (`mode = "node"`

), as well as the `windows`

argument, similarly to other “multiway” statistics implemented by tskit. See the relevant sections of the official *tskit* documentation for more on this topic.

Note that in the previous chunk we refered to individuals by their names (not numeric IDs of nodes as you would do it with *tskit* in Python). We allow this for readability and to make it easier to see which individuals are which based on the specified sampling schedule (the names are assigned to individuals based on the order of their sampling). We can get an overview of the individuals scheduled for sampling (i.e. permanently remembered) and their names with a helper function `ts_samples()`

:

`ts_samples(ts)`

```
#> # A tibble: 58 × 3
#> name time pop
#> <chr> <int> <chr>
#> 1 NEA_1 70000 NEA
#> 2 NEA_2 40000 NEA
#> 3 EUR_1 39170 EUR
#> 4 EUR_2 39134 EUR
#> 5 EUR_3 37738 EUR
#> 6 EUR_4 36530 EUR
#> 7 EUR_5 36362 EUR
#> 8 EUR_6 35944 EUR
#> 9 EUR_7 33900 EUR
#> 10 EUR_8 33853 EUR
#> # … with 48 more rows
```

That said, if you would like to run some statistics on nodes rather than on individuals, you can do it simply by using integer IDs instead of character names in each function’s interface.

Let’s try to put these new tools to practice and estimate the proportion of Neanderthal ancestry in Africans and Europeans in our simulated data. We can do this using the Patterson’s \(f_4\)-ratio statistic implemented in the `ts_f4ratio()`

function in *slendr* (you can find more information about this particular version of the statistic in this paper):

```
# first get a table of simulated African and European individuals in the tree-sequence
inds <- ts_samples(ts) %>% dplyr::filter(pop %in% c("AFR", "EUR"))
# estimate the amounts of Neanderthal ancestry in these individuals and add
# these values to the table
inds$ancestry <- ts_f4ratio(ts, X = inds$name, "NEA_1", "NEA_2", "AFR_1", "CH_1")$alpha
```

If we now summarise the inferred Neanderthal distribution in both populations, we see that there is no Neanderthal ancestry in Africans (as expected by our model–Africans did not receive a Neanderthal introgression pulse) but there is around 3% Neanderthal ancestry in Europeans (consistent with the 3% introgression pulse we simulated between 55000 and 50000 thousand years [ky] ago):

```
ggplot(inds, aes(pop, ancestry, fill = pop)) +
geom_boxplot() +
geom_jitter() +
labs(y = "Neanderthal ancestry proportion", x = "") +
theme(legend.position = "none") +
coord_cartesian(ylim = c(0, 0.1))
```

This is exactly as we specified in the model configuration above, suggesting that our simulations work as they should. You can see that there is quite a bit of noise but that’s because we simulated only 10 Mb of sequence.

We can also plot the trajectory of Neanderthal ancestry in Europe during the time-window for which we have simulated ancient and present-day DNA samples:

```
dplyr::filter(inds, pop == "EUR") %>%
ggplot(aes(time, ancestry)) +
geom_point() +
geom_smooth(method = "lm", linetype = 2, color = "red", size = 0.5) +
xlim(40000, 0) + coord_cartesian(ylim = c(0, 0.1)) +
labs(x = "time [years ago]", y = "Neanderthal ancestry proportion")
```

`#> `geom_smooth()` using formula 'y ~ x'`

Again, this is a result consistent with empirical estimates of Neanderthal ancestry using ancient DNA data.

In case you would like to verify some *f*-statistics results using the venerable ADMIXTOOLS software (see the linked paper which formally introduced these statistics in the first place), you can convert the tree-sequence data to a file format called EIGENSTRAT using the `ts_eigenstrat()`

function. The file conversion is internally handled by the R package *admixr* and returns an `EIGENSTRAT`

object which ties all individual `EIGENSTRAT`

file components together (see the tutorial to *admixr* for an extensive overview). *admixr* is an R package for running automated ADMIXTOOLS analyses entirely from R and makes these types of analyses very convenient.

`snps <- ts_eigenstrat(ts, prefix = file.path(tempdir(), "eigenstrat", "data"))`

`#> 121 multiallelic sites (0.208% out of 58089 total) detected and removed`

Running an *admixr* analysis is then as easy as plugging the object into an *admixr* function. For instance, we can estimate the proportion of Neanderthal ancestry in a couple of individuals \(X\) like this (*admixr* calls this proportion `alpha`

):

```
library(admixr)
f4ratio(data = snps, X = c("EUR_1", "EUR_2", "AFR_2"),
A = "NEA_1", B = "NEA_2", C = "AFR_1", O = "CH_1")
```

```
#> # A tibble: 3 × 8
#> A B X C O alpha stderr Zscore
#> <chr> <chr> <chr> <chr> <chr> <dbl> <dbl> <dbl>
#> 1 NEA_1 NEA_2 EUR_1 AFR_1 CH_1 0.0409 0.0374 1.10
#> 2 NEA_1 NEA_2 EUR_2 AFR_1 CH_1 0.00358 0.00374 0.956
#> 3 NEA_1 NEA_2 AFR_2 AFR_1 CH_1 0.000297 0.000962 0.308
```

In fact, lets compare the values obtained by both *tskit* and *admixr*/ADMIXTOOLS for all individuals:

```
europeans <- inds[inds$pop == "EUR", ]$name
# tskit result
result_ts <- ts_f4ratio(ts, X = europeans, A = "NEA_1", B = "NEA_2", C = "AFR_1", O = "CH_1") %>% dplyr::select(alpha_ts = alpha)
# result obtained by admixr/ADMIXTOOLS
result_admixr <- f4ratio(snps, X = europeans, A = "NEA_1", B = "NEA_2", C = "AFR_1", O = "CH_1") %>% dplyr::select(alpha_admixr = alpha)
dplyr::bind_cols(result_admixr, result_ts) %>%
ggplot(aes(alpha_ts, alpha_admixr)) +
geom_point() +
geom_abline(slope = 1, linetype = 2, color = "red", size = 0.5) +
labs(x = "f4-ratio statistic calculated with admixr/ADMIXTOOLS",
y = "f4-ratio statistic calculated with tskit")
```

The correspondence between the two looks good! 🎉 Again, note that the large amount of variance around the expected value of 3% ancestry is due to an extremely small data simulated here (only 10 Mb of sequence).

In case you need to process simulated data in some other software, you can use the function `ts_vcf()`

to save the simulated genotypes in a VCF format:

You can also specify only a subset of individuals to be saved in the VCF:

What follows is a very brief overview of other statistics which are implemented in *tskit* and for which *slendr* provides an easy-to-use R interface. As you will see, the goal of these functions is to get you to a result using a single function call, making them very convenient for quick interactive exploratory analyses on the simulated data right in the R console.

We will continue to use our simulated Neanderthal introgression tree-sequence data for these examples.

The \(F_{st}\) statistic is implemented by the function `ts_fst()`

.

If a single genome-wide \(F_{st}\) is to be calculated (i.e. not a window-based calculation), the `ts_fst()`

returns a simple three-column data frame

```
#> # A tibble: 1 × 3
#> x y Fst
#> <chr> <chr> <dbl>
#> 1 afr eur 0.0434
```

In case a non-named list of sample sets was provided, set names are generated automatically:

```
#> # A tibble: 1 × 3
#> x y Fst
#> <chr> <chr> <dbl>
#> 1 set_1 set_2 0.0434
```

Of course, this is much less readable and we encourage you to name the sample sets appropriately.

In case more than two sample sets are specified, all pairwise statistics are computed:

```
ts_fst(ts, sample_sets = list(afr = c("AFR_1", "AFR_2", "AFR_3"),
eur = c("EUR_1", "EUR_2"),
nea = c("NEA_1", "NEA_2")))
```

```
#> # A tibble: 3 × 3
#> x y Fst
#> <chr> <chr> <dbl>
#> 1 afr eur 0.0434
#> 2 afr nea 0.430
#> 3 eur nea 0.424
```

As with many other statistics implemented by *tskit**,* `ts_fst()`

accepts a `windows`

argument, specifying the breakpoints between windows. In this case, the `Fst`

column in the resulting data frame is a so called “list-column”, with each item in the column being a vector of \(F_{st}\) values, one per each window. List-columns can be a little confusing for new R users, but we highly encourage you to get used to them as they allow extremely consise and elegant handling of structured data within normal data frames (you can start with this introduction).

```
# define breakpoints between 20 windows
breakpoints <- seq(0, ts$sequence_length, length.out = 21)
# calculate window-based Fst statistic
win_fst <- ts_fst(
ts, windows = breakpoints,
sample_sets = list(afr = c("AFR_1", "AFR_2", "AFR_3"),
eur = c("EUR_1", "EUR_2"),
nea = c("NEA_1", "NEA_2"))
)
# we get 20 values for each parwise calculation
win_fst
```

```
#> # A tibble: 3 × 3
#> x y Fst
#> <chr> <chr> <named list>
#> 1 afr eur <dbl [20]>
#> 2 afr nea <dbl [20]>
#> 3 eur nea <dbl [20]>
```

For instance, here are window-based \(F_st\) values for the `afr-vs-eur`

calculation (first row of the table above):

`win_fst[1, ]$Fst`

```
#> $`1`
#> [1] 0.033291095 0.016539440 0.056243368 0.056683472 0.041666667
#> [6] 0.041250832 0.025904371 0.037193831 0.015493634 0.014280070
#> [11] 0.068641714 0.027763295 -0.002557545 0.012265614 0.031870265
#> [16] 0.030547550 0.118254879 0.081014980 0.079867987 0.109555032
```

The function `ts_tajima()`

has nearly the same interface as `ts_fst()`

shown above.

If a non-window version is to be calculated, we get a single genome-wide values for each sample set (named or non-named list of character vectors with individual names):

```
#> # A tibble: 2 × 2
#> set D
#> <chr> <dbl>
#> 1 afr -0.223
#> 2 eur -0.106
```

For window-based version, the function returns the `D`

column as a list column of vectors with \(i\)-th element being the Tajima’s D value for the \(i\)-th window:

```
#> # A tibble: 2 × 2
#> set D
#> <chr> <named list>
#> 1 afr <dbl [20]>
#> 2 eur <dbl [20]>
```

We can calculate diversity within given groups of individuals with the function `ts_diversity()`

. For instance, even in our extremely simplified example, we would expect the highest levels of diversity in Africans, followed by Europeans, Neanderthals and the “degenerate” single individual outgroup “chimpanzee”. Is this true? Let’s find out.

First we extract individuals from all populations, creating a list of character vectors for each group (which is what functions such as `ts_diversity()`

expects as an input):

```
# get sampled individuals from all populations
sample_sets <- ts_samples(ts) %>%
split(., .$pop) %>%
lapply(function(pop) pop$name)
sample_sets
```

```
#> $AFR
#> [1] "AFR_1" "AFR_2" "AFR_3" "AFR_4" "AFR_5"
#>
#> $CH
#> [1] "CH_1"
#>
#> $EUR
#> [1] "EUR_1" "EUR_2" "EUR_3" "EUR_4" "EUR_5" "EUR_6" "EUR_7" "EUR_8"
#> [9] "EUR_9" "EUR_10" "EUR_11" "EUR_12" "EUR_13" "EUR_14" "EUR_15" "EUR_16"
#> [17] "EUR_17" "EUR_18" "EUR_19" "EUR_20" "EUR_21" "EUR_22" "EUR_23" "EUR_24"
#> [25] "EUR_25" "EUR_27" "EUR_26" "EUR_28" "EUR_29" "EUR_30" "EUR_31" "EUR_32"
#> [33] "EUR_33" "EUR_34" "EUR_35" "EUR_36" "EUR_37" "EUR_38" "EUR_39" "EUR_40"
#> [41] "EUR_41" "EUR_42" "EUR_43" "EUR_44" "EUR_45" "EUR_46" "EUR_47" "EUR_48"
#> [49] "EUR_49" "EUR_50"
#>
#> $NEA
#> [1] "NEA_1" "NEA_2"
```

Now we can calculate diversity in each population and sort the results in an increasing order of diversity:

`ts_diversity(ts, sample_sets) %>% dplyr::arrange(diversity)`

```
#> # A tibble: 4 × 2
#> set diversity
#> <chr> <dbl>
#> 1 CH 0.0000001
#> 2 NEA 0.0000460
#> 3 EUR 0.000243
#> 4 AFR 0.000253
```

Great! This matches our expectations. We simulated chimp “population” as only one individual, so we expect essentially no diversity after millions of years of evolution.

We can calculate pairwise divergence between groups of individuals using the function `ts_divergence()`

. Given our model, we would expect the lowest divergence between the two modern human groups `AFR`

and `EUR`

, then between Neanderthals and the two modern humans, and all three groups (`AFR`

, `EUR`

and `NEA`

) should have equal, much deeper divergence from the outgroup chimpanzee `CH`

.

`ts_divergence(ts, sample_sets) %>% dplyr::arrange(divergence)`

```
#> # A tibble: 6 × 3
#> x y divergence
#> <chr> <chr> <dbl>
#> 1 AFR EUR 0.000279
#> 2 EUR NEA 0.000368
#> 3 AFR NEA 0.000384
#> 4 CH NEA 0.00396
#> 5 CH EUR 0.00397
#> 6 AFR CH 0.00398
```

After sorting the table based on the value in the divergence column, we can see the results fit our expectations.

These were only a couple of examples of statistical functions implemented in *tskit* for which we provide a native R interface in *slendr*. You can find more tree-sequence statistics in the reference manual on the project website. Not all statistics from the *tskit* library are implemented, but we intend to expand the selection provided by *slendr* in the near future. If there is some functionality that you would like to use in your project missing in *slendr*, please don’t hesitate to let us now by creating an issue on our GitHub page.