This tutorial estimates the rate of evolution from a set of virus sequences which have been isolated at different points in time (heterochronous or time-stamped data). The data are 129 sequences from the G (attachment protein) gene of human respiratory syncytial virus subgroup A (RSVA) from various parts of the world with isolation dates ranging from 1956-2002 ((Zlateva, Lemey, Vandamme, & Van Ranst, 2004), (Zlateva, Lemey, Moës, Vandamme, & Van Ranst, 2005)). RSVA causes infections of the lower respiratory tract causing symptoms that are often indistinguishable from the common cold. By age 3, nearly all children will be infected and a small percentage (<3%) will develop more serious inflammation of the bronchioles requiring hospitalisation.
The aim of this tutorial is to obtain estimates for :
the rate of molecular evolution
the date of the most recent common ancestor
the phylogenetic relationships with measures of statistical support.
The following software will be used in this tutorial:
BEAST - this package contains the BEAST program, BEAUti, DensiTree, TreeAnnotator and other utility programs. This tutorial is written for BEAST v
2.5.x, which has support for multiple partitions. It is available for download from\ http://www.beast2.org.
Tracer - this program is used to explore the output of BEAST (and other Bayesian MCMC programs). It graphically and quantitively summarises the distributions of continuous parameters and provides diagnostic information. At the time of writing, the current version is v
1.7. It is available for download from http://beast.community/tracer.
FigTree - this is an application for displaying and printing molecular phylogenies, in particular those obtained using BEAST. At the time of writing, the current version is v
1.4.3. It is available for download from http://beast.community/figtree.
The NEXUS alignment
The data is in a file called
You can find it in the
examples/nexus directory in the directory
where BEAST was installed. Or click the link to download the data. After
the data is opened in your web browser, right click mouse and save it as
This file contains an alignment of 129 sequences from the G gene of RSVA
virus, 629 nucleotides in length. Import this alignment into BEAUti.
Because this is a protein-coding gene we are going to split the
alignment into three partitions representing each of the three codon
positions. To do this we will click the
Split button at the
bottom of the
Partitions panel and then select the
1 + 2 + 3
frame 3 from the drop-down menu (Figure [fig:BEAUti_split]).
This signifies that the first full codon starts at the third nucleotide
in the alignment. This will create three rows in the partitions panel.
You will have to re-link the tree and clock models across the three
partitions (and name them
clock respectively) before
continuing to the next step. The partition panel should now look
something like this:
By default all the taxa are assumed to have a date of zero (i.e. the
sequences are assumed to be sampled at the same time). In this case, the
RSVA sequences have been sampled at various dates going back to the
1950s. The actual year of sampling is given in the name of each taxon
and we could simply edit the value in the Date column of the table to
reflect these. However, if the taxa names contain the calibration
information, then a convenient way to specify the dates of the sequences
in BEAUti is to click the checkbox
Use tip dates and then use
Configure button at the top of the
Tip Dates panel.
Clicking this will make a dialog box appear.
Select the option to
use everything, choose
after last from from
drop-down box and type
s into the corresponding text box. This will
extract the trailing numbers from the taxon names after the last little
s, which are interpreted as the year (in this case since 1900) that
the sample was isolated.
The dates panel should now look something like this:
Setting the substitution model
We will use the HKY model with empirical base frequencies for all three
partitions. To do this first link the site partitions and then choose
HKY and Empirical from the Subst Model and Frequencies drop-boxes. Also
check the estimate box for the Mutation Rate,which will finally trigger
to check the
Fix mean mutation rate box.
shift key to select all site models on the left side, and
OK to clone the setting from defined site model (Figure
[fig:cloneFrom]). Go through each site model, as you can see, their
configurations are same now.
We are going to use the strict clock model, which is the default, so no changes are necessary in the clock model panel.
To set up the priors, select the
Priors tab. Choose
Coalescent Constant Population
for the tree prior. Set the prior on the clockRate
parameter to a log-normal with
Setting the MCMC options
For this dataset let’s initially set the chain length to
1000000 as this will
run reasonably quickly on most modern computers. Set the sampling
frequencies for the screen to
10000, the trace log file to
400 and the trees
Save the BEAST file (e.g.
RSV2.xml) and run it in BEAST.
Now run BEAST and when it asks for an input file, provide your newly created XML file as input. BEAST will then run until it has finished reporting information to the screen. The actual results files are save to the disk in the same location as your input file. The output to the screen will look something like this:
BEAST v2.5.2, 2002-2019 Bayesian Evolutionary Analysis Sampling Trees Designed and developed by Remco Bouckaert, Alexei J. Drummond, Andrew Rambaut & Marc A. Suchard Department of Computer Science University of Auckland email@example.com firstname.lastname@example.org Institute of Evolutionary Biology University of Edinburgh email@example.com David Geffen School of Medicine University of California, Los Angeles firstname.lastname@example.org Downloads, Help & Resources: http://beast2.org/ Source code distributed under the GNU Lesser General Public License: http://github.com/CompEvol/beast2 BEAST developers: Alex Alekseyenko, Trevor Bedford, Erik Bloomquist, Joseph Heled, Sebastian Hoehna, Denise Kuehnert, Philippe Lemey, Wai Lok Sibon Li, Gerton Lunter, Sidney Markowitz, Vladimir Minin, Michael Defoin Platel, Oliver Pybus, Tim Vaughan, Chieh-Hsi Wu, Walter Xie Thanks to: Roald Forsberg, Beth Shapiro and Korbinian Strimmer ... ... 990000 -6108.0939 -5503.4454 -604.6484 1m45s/Msamples 1000000 -6102.6691 -5505.1198 -597.5493 1m44s/Msamples Operator Tuning #accept #reject Pr(m) Pr(acc|m) ScaleOperator(StrictClockRateScaler.c:clock) 0.78157 8218 27756 0.03601 0.22844 UpDownOperator(strictClockUpDownOperator.c:clock) 0.84475 551 35258 0.03601 0.01539 Try setting scaleFactor to about 0.919 ScaleOperator(KappaScaler.s:RSV2_1) 0.38916 323 934 0.00120 0.25696 DeltaExchangeOperator(FixMeanMutationRatesOperator) 0.33840 4766 19363 0.02401 0.19752 ScaleOperator(KappaScaler.s:RSV2_2) 0.39201 288 901 0.00120 0.24222 ScaleOperator(KappaScaler.s:RSV2_3) 0.41649 271 964 0.00120 0.21943 ScaleOperator(CoalescentConstantTreeScaler.t:tree) 0.71887 273 35495 0.03601 0.00763 Try setting scaleFactor to about 0.848 ScaleOperator(CoalescentConstantTreeRootScaler.t:tree) 0.64576 3140 33308 0.03601 0.08615 Try setting scaleFactor to about 0.804 Uniform(CoalescentConstantUniformOperator.t:tree) - 193184 166770 0.36014 0.53669 SubtreeSlide(CoalescentConstantSubtreeSlide.t:tree) 4.11043 28087 152489 0.18007 0.15554 Exchange(CoalescentConstantNarrow.t:tree) - 44602 135445 0.18007 0.24772 Exchange(CoalescentConstantWide.t:tree) - 84 35705 0.03601 0.00235 WilsonBalding(CoalescentConstantWilsonBalding.t:tree) - 205 35530 0.03601 0.00574 ScaleOperator(PopSizeScaler.t:tree) 0.60390 10084 26007 0.03601 0.27940 Tuning: The value of the operator's tuning parameter, or '-' if the operator can't be optimized. #accept: The total number of times a proposal by this operator has been accepted. #reject: The total number of times a proposal by this operator has been rejected. Pr(m): The probability this operator is chosen in a step of the MCMC (i.e. the normalized weight). Pr(acc|m): The acceptance probability (#accept as a fraction of the total proposals for this operator). Total calculation time: 106.096 seconds End likelihood: -6102.669168760964
Analysing the BEAST output
Note that the effective sample sizes (ESSs) for many of the logged quantities are small (ESSs less than 100 will be highlighted in red by Tracer). This is not good. A low ESS means that the trace contains a lot of correlated samples and thus may not represent the posterior distribution well. In the bottom right of the window is a frequency plot of the samples which is expected given the low ESSs is extremely rough.
If we select the tab on the right-hand-side labelled
Trace we can view
the raw trace, that is, the sampled values against the step in the MCMC
Here you can see how the samples are correlated. There are 2500 samples
in the trace (we ran the MCMC for steps sampling every 400) but adjacent
samples often tend to have similar values. The ESS for the absolute rate
of evolution (clockRate) is about
65 so we are only getting 1
independent sample to every
65 ~ 2500/38 actual samples). With a short
run such as this one, it may also be the case that the default burn-in
of 10% of the chain length is inadequate. Not excluding enough of the
start of the chain as burn-in will render estimates of ESS unreliable.
The simple response to this situation is that we need to run the chain
for longer. Given the lowest ESS (for the constant coalescent) is
50, it would suggest that we have to run the chain for at least
4 times the length to get reasonable ESSs that are
>200. So let’s go
for a chain length of 6000000 and log every 5000. Go back to the
options section in BEAUti, and create a new BEAST XML file with a longer
chain length. Now run BEAST and load the new log file into Tracer (you
can leave the old one loaded for comparison).
Click on the Trace tab and look at the raw trace plot.
We have chosen options that produce 12000 samples and with an ESS
239 there is still auto-correlation between the samples but
>239 effectively independent samples will now provide a very good
estimate of the posterior distribution. There are no obvious trends in
the plot which would suggest that the MCMC has not yet converged, and
there are no significant long range fluctuations in the trace which
would suggest poor mixing.
As we are satisfied with the mixing we can now move on to one of the
parameters of interest: substitution rate. Select
clockRate in the
left-hand table. This is the average substitution rate across all sites
in the alignment. Now choose the density plot by selecting the tab
Marginal Density. This shows a plot of the marginal
posterior probability density of this parameter. You should see a plot
similar to this:
As you can see the posterior probability density is roughly bell-shaped.
There is some sampling noise which would be reduced if we ran the chain
for longer or sampled more often but we already have a good estimate of
the mean and HPD interval. You can overlay the density plots of multiple
traces in order to compare them (it is up to the user to determine
whether they are comparable on the the same axis or not). Select the
relative substitution rates for all three codon positions in the table
to the left (labelled
mutationRate.3). You will now see the posterior probability densities
for the relative substitution rate at all three codon positions
Summarising the trees
Use the program TreeAnnotator to summarise the tree. TreeAnnotator is an application that comes with BEAST.
Summary trees can be viewed using FigTree (a program separate from BEAST) and DensiTree (distributed with BEAST).
Below a DensiTree with clade height bars for clades with over 50% support. Root canal tree represents maximum clade credibility tree.
In what year did the common ancestor of all RSVA viruses sampled live? What is the 95% HPD?
Bonus section: Bayesian Skyline plot
We can reconstruct the population history using the Bayesian Skyline
plot. In order to do so, load the XML file into BEAUti, select the
priors-tab and change the tree prior from coalescent with constant
population size to coalescent with Bayesian skyline. Note that an extra
item is added to the priors called
Markov chained population sizes
which is a prior that ensures dependence between population sizes.
By default the number of groups used in the skyline analysis is set to
5, To change this, select menu View/Show Initialization panel and a list
of parameters is shown. Select
bPopSizes.t:tree and change the
dimension to 3. Likewise, selection
bGroupSizes.t:tree and change
its dimension to 3. The dimensions of the two parameters should be the
same. More groups mean more population changes can be detected, but it
also means more parameters need to be estimated and the chain runs
longer. The extended Bayesian skyline plot automatically detects the
number of changes, so it could be used as an alternative tree prior.
This analysis requires a bit longer to converge, so change the MCMC
chain length to 10 million, and the log intervals for the trace-log and
tree-log to 10 thousand. Then, save the file and run BEAST. You can also
download the log (
RSV2-bsp.log) and tree (
tree-bsp.trees) files from
To plot the population history, load the log file in tracer and select the menu Analysis/Bayesian Skyline Reconstruction.
A dialog is shown where you can specify the tree file associated with the log file. Also, since the youngest sample is from 2002, change the entry for age of youngest tip to 2002.
After some calculation, a graph appears showing population history where
the median and 95% HPD intervals are plotted. After selecting the
solid interval checkbox, the graph should look something like this.
By what amount did the effective population size of RSVA grow from 1970 to 2002 according to the BSP?
What are the underlying assumptions of the BSP? Are the violated by this data set?
Change the Bayesian skyline prior to extended Bayesian skyline plot
(EBSP) prior and run till convergence. EBSP produces an extra log file,
$(seed) is replaced by the seed you used
to run BEAST. A plot can be created by running the EBSPAnalyser utility,
and loading the output file in a spreadsheet.
How many groups are indicated by the EBSP analysis? This is much lower than for BSP. How does this affect the population history plots?
- Bayesian Evolutionary Analysis with BEAST 2 (Drummond & Bouckaert, 2014)
- BEAST 2 website and documentation: http://www.beast2.org/
- Join the BEAST user discussion: http://groups.google.com/group/beast-users
- Zlateva, K. T., Lemey, P., Vandamme, A.-M., & Van Ranst, M. (2004). Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein. J Virol, 78(9), 4675–4683.
- Zlateva, K. T., Lemey, P., Moës, E., Vandamme, A.-M., & Van Ranst, M. (2005). Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Virol, 79(14), 9157–9167. https://doi.org/10.1128/JVI.79.14.9157-9167.2005
- Drummond, A. J., & Bouckaert, R. R. (2014). Bayesian evolutionary analysis with BEAST 2. Cambridge University Press.
If you found Taming the BEAST helpful in designing your research, please cite the following paper:
Joëlle Barido-Sottani, Veronika Bošková, Louis du Plessis, Denise Kühnert, Carsten Magnus, Venelin Mitov, Nicola F. Müller, Jūlija Pečerska, David A. Rasmussen, Chi Zhang, Alexei J. Drummond, Tracy A. Heath, Oliver G. Pybus, Timothy G. Vaughan, Tanja Stadler (2018). Taming the BEAST – A community teaching material resource for BEAST 2. Systematic Biology, 67(1), 170–-174. doi: 10.1093/sysbio/syx060