Category: linear models

Analyzing a simple experiment with heterogeneous variances using asreml, MCMCglmm and SAS

I was working with a small experiment which includes families from two Eucalyptus species and thought it would be nice to code a first analysis using alternative approaches. The experiment is a randomized complete block design, with species as fixed effect and family and block as a random effects, while the response variable is growth strain (in \( mu epsilon\)).

When looking at the trees one can see that the residual variances will be very different. In addition, the trees were growing in plastic bags laid out in rows (the blocks) and columns. Given that trees were growing in bags siting on flat terrain, most likely the row effects are zero.

Below is the code for a first go in R (using both MCMCglmm and ASReml-R) and SAS. I had stopped using SAS for several years, mostly because I was running a mac for which there is no version. However, a few weeks ago I started accessing it via their OnDemand for Academics program via a web browser.

The R code using REML looks like:

While using MCMC we get estimates in the ballpark by using:

The SAS code is not that disimilar, except for the clear demarcation between data processing (data step, for reading files, data transformations, etc) and specific procs (procedures), in this case to summarize data, produce a boxplot and fit a mixed model.

SAS boxplot for the data set.
SAS boxplot for the data set.

I like working with multiple languages and I realized that, in fact, I missed SAS a bit. It was like meeting an old friend; at the beginning felt strange but we were quickly chatting away after a few minutes.

More sense of random effects

I can’t exactly remember how I arrived to Making sense of random effects, a good post in the Distributed Ecology blog (go over there and read it). Incidentally, my working theory is that I follow Scott Chamberlain (@recology_), who follows Karthik Ram ?(@_inundata) who mentioned Edmund Hart’s (@DistribEcology) post. I liked the discussion, but I thought one could add to the explanation to make it a bit clearer.

The idea is that there are 9 individuals, assessed five times each—once under each of five different levels for a treatment—so we need to include individual as a random effect; after all, it is our experimental unit. The code to generate the data, plot it and fit the model is available in the post, but I redid data generation to make it a bit more R-ish and, dare I say, a tad more elegant:

Original simulated data, showing the effect of treatment (fixed effect) and each individual.

What we can do to better understand what’s going on is ‘adjust’ the score observations by the estimated fixed effects and plot those values to see what we are modeling with the random effects:

Data ‘adjusted’ by fixed effects. The random intercepts would be lines going through the average of points for each individual.

The random effects for individual or, better, the individual-level intercepts are pretty much the lines going through the middle of the points for each individual. Furthermore, the variance for ind is the variance of the random intercepts around the’adjusted’ values, which can be seen comparing the variance of random effects above (~7.15) with the result below (~7.13).

Distributed Ecology then goes on to randomize randomize the individuals within treatment, which means that the average deviation around the adjusted means is pretty close to zero, making that variance component close to zero. I hope this explanation complements Edmund Hart’s nice post.

P.S. If you happen to be in the Southern part of South America next week, I’ll be here and we can have a chat (and a beer, of course).

Overlay of design matrices in genetic analysis

I’ve ignored my quantitative geneticist side of things for a while (at least in this blog) so this time I’ll cover some code I was exchanging with a couple of colleagues who work for other organizations.

It is common to use diallel mating designs in plant and tree breeding, where a small number of parents acts as both males and females. For example, with 5 parents we can have 25 crosses, including reciprocals and selfing (crossing an individual with itself). Decades ago this mating design was tricky to fit and, considering an experimental layout with randomized complete blocks, one would have something like y = mu + blocks + dads + mums + cross + error. In this model dads and mums were estimating a fraction of the additive genetic variance. With the advent of animal model BLUP, was possible to fit something like y = mu + blocks + individual (using a pedigree) + cross + error. Another less computationally demanding alternative (at least with unrelated parents) is to fit a parental model, overlaying the design matrices for parents with something like this y = mu + blocks + (dad + mum) + cross + error.

The following code simulate data for a a diallel trial with three replicates and runs a parental model with ASReml. Later I expand the analysis building the matrices by hand.

How is the matrix overlay working? We can replicate the calculations used by ASReml by building the matrices from scratch and reusing the variance components, so we avoid the nastiness of writing code for residual maximum likelihood. Once I build the basic matrices I use the code from my How does a linear mixed model look like? post.

The overlay matrix Zparents is double the actual value we should use:

Repeating the analyses ‘by hand’ using Zparents2 to build the Z matrix results in the same overall mean and block effects, but the predicted breeding values for parents when using Zparents are 0.7 of the predicted breeding values for parents when using Zparents2. I may need to refit the model and obtain new variance components for parents when working with the correct overlaid matrix.

Gratuitous picture: walking in Quail Island (Photo: Luis).

INLA: Bayes goes to Norway

INLA is not the Norwegian answer to ABBA; that would probably be a-ha. INLA is the answer to ‘Why do I have enough time to cook a three-course meal while running MCMC analyses?”.

Integrated Nested Laplace Approximations (INLA) is based on direct numerical integration (rather than simulation as in MCMC) which, according to people ‘in the know’, allows:

  • the estimation of marginal posteriors for all parameters,
  • marginal posteriors for each random effect and
  • estimation of the posterior for linear combinations of random effects.

Rather than going to the usual univariate randomized complete block or split-plot designs that I have analyzed before (here using REML and here using MCMC), I’ll go for some analyses that motivated me to look for INLA. I was having a look at some reproductive output for Drosophila data here at the university, and wanted to fit a logistic model using MCMCglmm. Unfortunately, I was running into the millions (~3M) of iterations to get a good idea of the posterior and, therefore, leaving the computer running overnight. Almost by accident I came across INLA and started playing with it. The idea is that Sol—a Ph.D. student—had a cool experiment with a bunch of flies using different mating strategies over several generations, to check the effect on breeding success. Therefore we have to keep track of the pedigree too.

Gratuitous picture: Cubist apartments not in Norway (Photo: Luis, click to enlarge).

Up to this point we have read the response data, the pedigree and constructed the inverse of the pedigree matrix. We also needed to build a contrast matrix to compare the mean response between the different mating strategies. I was struggling there and contacted Gregor Gorjanc, who kindly emailed me the proper way to do it.

There is another related package (Animal INLA) that takes care of i- giving details about the priors and ii- “easily” fitting models that include a term with a pedigree (an animal model in quantitative genetics speak). However, I wanted the assumptions to be clear so read the source of Animal INLA and shamelessly copied the useful bits (read the source, Luke!).

A quick look at the time taken by INLA shows that it is in the order of seconds (versus overnight using MCMC). I have tried a few examples and the MCMCglmm and INLA results tend to be very close; however, figuring out how to code models has been very tricky for me. INLA follows the glorious tradition of not having a ‘proper’ manual, but a number of examples with code. In fact, they reimplement BUGS‘s examples. Personally, I struggle with that approach towards documentation, but you may be the right type of person for that. Note for letter to Santa: real documentation for INLA.

I was talking with a student about using Norwegian software and he mentioned Norwegian Black Metal. That got me thinking about how the developers of the package would look like; would they look like Gaahl of Gorgoroth (see interview here)?

Gaahl Gorgoroth
Not an INLA developer

Talk about disappointment! In fact Håvard Rue, INLA mastermind, looks like a nice, clean, non-black-metal statistician. To be fair, it would be quite hard to code in any language wearing those spikes…

Split-plot 2: let’s throw in some spatial effects

Disappeared for a while collecting frequent flyer points. In the process I ‘discovered’ that I live in the middle of nowhere, as it took me 36 hours to reach my conference destination (Estoril, Portugal) through Christchurch, Sydney, Bangkok, Dubai, Madrid and Lisbon.

Where was I? Showing how split-plots look like under the bonnet (hood for you US readers). Yates presented a nice diagram of his oats data set in the paper, so we have the spatial location of each data point which permits us playing with within-trial spatial trends.

Rather than mucking around with typing coordinates we can rely on Kevin Wright’s version of the oats dataset contained in the agridat package. Kevin is a man of mistery, a James Bond of statisticians—so he keeps a low profile—with a keen interest in experimental design and analyses. This chap has put a really nice collection of data sets WITH suggested coding for the analyses, including nlme, lme4, asreml, MCMCglmm and a few other bits and pieces. Recommended!

Plants (triffids excepted) do not move, which means that environmental trends within a trial (things like fertility, water availability, temperature, etc) can affect experimental units in a way that varies with space and which induces correlation of the residuals. Kind of we could be violating the independence assumption if you haven’t got the hint yet.

Gratuitous picture: Detail of Mosteiro dos Jerónimos, Belém, Lisboa, Portugal (Photo: Luis).

There are a few ways to model environmental trends (AR processes, simple polynomials, splines, etc) that can be accounted for either through the G matrix (as random effects) or the R matrix. See previous post for explanation of the bits and pieces. We will use here a very popular approach, which is to consider two separable (so we can estimate the bloody things) autoregressive processes, one for rows and one for columns, to model spatial association. In addition, we will have a spatial residual. In summary, the residuals have moved from \( oldsymbol{R} = sigma^2_e oldsymbol{I}\) to \( oldsymbol{R} = sigma^2_s oldsymbol{R}_{col} otimes oldsymbol{R}_{row}\). I previously showed the general form of this autoregressive matrices in this post, and you can see the \( oldsymbol{R}_{col}\) matrix below. In some cases we can also add an independent residual (the so-called nugget) to the residual matrix.

We will first fit a split-plot model considering spatial residuals using asreml because, let’s face it, there is no other package that will give you the flexibility:

So we have to build an autoregressive correlation matrix for rows, one for columns and multiply the whole thing for a spatial variance. Then we can add an independent residual (the nugget, if we want—and can estimate—one). Peter Dalgaard has neat code for building the autocorrelation matrix. And going back to the code in the previous post:

Which are the same results one gets from ASReml-R. QED.

P.S. Many thanks to Kevin Wright for answering my questions about agridat.

Split-plot 1: How does a linear mixed model look like?

I like statistics and I struggle with statistics. Often times I get frustrated when I don’t understand and I really struggled to make sense of Krushke’s Bayesian analysis of a split-plot, particularly because ‘it didn’t look like’ a split-plot to me.

Additionally, I have made a few posts discussing linear mixed models using several different packages to fit them. At no point I have shown what are the calculations behind the scenes. So, I decided to combine my frustration and an explanation to myself in a couple of posts. This is number one and the follow up is Split-plot 2: let’s throw in some spatial effects.

Example of forestry split-plot: one of my colleagues has a trial in which stocking (number of trees per ha) is the main plot and fertilization is the subplot (higher stockings look darker because trees are closer together).

How do linear mixed models look like

Linear mixed models, models that combine so-called fixed and random effects, are often represented using matrix notation as:

\( oldsymbol{y} = oldsymbol{X b} + oldsymbol{Z a} + oldsymbol{e}\)

where \( oldsymbol{y}\) represents the response variable vector, \( oldsymbol{X} mbox{ and } oldsymbol{Z}\) are incidence matrices relating the response to the fixed (\( oldsymbol{b}\) e.g. overall mean, treatment effects, etc) and random (\( oldsymbol{a}\), e.g. family effects, additive genetic effects and a host of experimental design effects like blocks, rows, columns, plots, etc), and last the random residuals (\( oldsymbol{e}\)).

The previous model equation still requires some assumptions about the distribution of the random effects. A very common set of assumptions is to say that the residuals are iid (identical and independently distributed) normal (so \( oldsymbol{R} = sigma^2_e oldsymbol{I} \)) and that the random effects are independent of each other, so \( oldsymbol{G} = oldsymbol{B} igoplus oldsymbol{M}\) is a direct sum of the variance of blocks (\( oldsymbol{B} = sigma^2_B oldsymbol{I}\)) and main plots (\( oldsymbol{M} = sigma^2_M oldsymbol{I}\)).

An interesting feature of this matrix formulation is that we can express all sort of models by choosing different assumptions for our covariance matrices (using different covariance structures). Do you have longitudinal data (units assessed repeated times)? Is there spatial correlation? Account for this in the \( oldsymbol{R}\) matrix. Random effects are correlated (e.g. maternal and additive genetic effects in genetics)? Account for this in the \( oldsymbol{G}\) matrix. Multivariate response? Deal with unstructured \( oldsymbol{R}\) and \( oldsymbol{G}\), or model the correlation structure using different constraints (and the you’ll need asreml).

By the way, the history of linear mixed models is strongly related to applications of quantitative genetics for the prediction of breeding values, particularly in dairy cattle. Charles Henderson developed what is now called Henderson’s Mixed Model Equations† to simultaneously estimate fixed effects and predict random genetic effects:

\(
left [
egin{array}{cc}
oldsymbol{X}’ oldsymbol{R}^{-1} oldsymbol{X} & oldsymbol{X}’ oldsymbol{R}^{-1} oldsymbol{Z} \
oldsymbol{Z}’ oldsymbol{R}^{-1} oldsymbol{X} & oldsymbol{Z}’ oldsymbol{R}^{-1} oldsymbol{Z} + oldsymbol{G}^{-1}
end{array}

ight ]
left [
egin{array}{c}
oldsymbol{b} \
oldsymbol{a}
end{array}

ight ] =
left [
egin{array}{c}
oldsymbol{X}’ oldsymbol{R}^{-1} oldsymbol{y} \
oldsymbol{Z}’ oldsymbol{R}^{-1} oldsymbol{y}
end{array}

ight ] \)

The big matrix on the left-hand side of this equation is often called the \( oldsymbol{C} \) matrix. You could be thinking ‘What does this all mean?’ It is easier to see what is going on with a small example, but rather than starting with, say, a complete block design, we’ll go for a split-plot to start tackling my annoyance with the aforementioned blog post.

Old school: physical split-plots

Given that I’m an unsophisticated forester and that I’d like to use data available to anyone, I’ll rely on an agricultural example (so plots are actually physical plots in the field) that goes back to Frank Yates. There are two factors (oats variety, with three levels, and fertilization, with four levels). Yates, F. (1935) Complex experiments, Journal of the Royal Statistical Society Suppl. 2, 181–247 (behind pay wall here).

Layout of oats experiment in Yates’s paper, from a time when articles were meaty. Each of the 6 replicates is divided in to 3 main plots for oats varieties (v1, v2 and v3), while each variety was divided into four parts with different levels of fertilization (manure—animal crap—n1 to n4). Cells display yield.

Now it is time to roll up our sleeves and use some code, getting the data and fitting the same model using nlme (m1) and asreml (m2), just for the fun of it. Anyway, nlme and asreml produce exactly the same results.

We will use the oats data set that comes with MASS, although there is also an Oats data set in nlme and another version in the asreml package. (By the way, you can see a very good explanation by Bill Venables of a ‘traditional’ ANOVA analysis for a split-plot here):

Now we can move to implement the Mixed Model Equations, where probably the only gotcha is the definition of the \(oldsymbol{Z}\) matrix (incidence matrix for random effects), as both nlme and asreml use ‘number of levels of the factor’ for both the main and interactions effects, which involves using the contrasts.arg argument in model.matrix().

Not surprisingly, we get the same results, except that we start assuming the variance components from the previous analyses, so we can avoid implementing the code for restricted maximum likelihood estimation as well. By the way, given that \( oldsymbol{R}^{-1} \) is in all terms it can be factored out from the MME, leaving terms like \( oldsymbol{X}’ oldsymbol{X} \), i.e. without \( oldsymbol{R}^{-1}\), making for simpler calculations. In fact, if you drop the \( oldsymbol{R}^{-1} \) it is easier to see what is going on in the different components of the \( oldsymbol{C} \) matrix. For example, print \( oldsymbol{X}’ oldsymbol{X} \) and you’ll get the sum of observations for the overall mean and for each of the levels of the fixed effect factors. Give it a try with the other submatrices too!

I will leave it here and come back to this problem as soon as I can.

† Incidentally, a lot of the theoretical development was supported by Shayle Searle (a Kiwi statistician and Henderson’s colleague in Cornell University).

Bivariate linear mixed models using ASReml-R with multiple cores

A while ago I wanted to run a quantitative genetic analysis where the performance of genotypes in each site was considered as a different trait. If you think about it, with 70 sites and thousands of genotypes one is trying to fit a 70×70 additive genetic covariance matrix, which requires 70*69/2 = 2,415 covariance components. Besides requiring huge amounts of memory and being subject to all sort of estimation problems there were all sort of connectedness issues that precluded the use of Factor Analytic models to model the covariance matrix. The best next thing was to run over 2,000 bivariate analyses to build a large genetic correlation matrix (which has all sort of issues, I know). This meant leaving the computer running for over a week.

In another unrelated post, Kevin asked if I have ever considered using ASReml-R to run in parallel using a computer with multiple cores. Short answer, I haven’t, but there is always a first time.

The code runs in my laptop (only two cores) but I still have to test its performance in my desktop (4 cores) and see if it really makes a difference in time versus running the analyses sequentially. Initially my mistake was using the multicore package (require(multicore)) directly, which doesn’t start the ‘parallel backend’ and ran sequentially. Using require(doMC) loads multicore but takes care of starting the ‘parallel backend’.

Gratuitous picture: Trees at 8 pm illuminated by bonfire and full moon (Photo: Luis).

P.S. Thanks to Etienne Laliberté, who sent me an email in early 2010 pointing out that I had to use doMC. One of the few exceptions in the pile of useless emails I have archived.
P.S.2. If you want to learn more about this type of models I recommend two books: Mrode’s Linear Models for the Prediction of Animal Breeding Values, which covers multivariate evaluation with lots of gory details, and Lynch and Walsh’s Genetics and Analysis of Quantitative Traits, which is the closest thing to the bible in quantitative genetics.
P.S.3. 2012-05-08. I did run the code in my desktop (iMac with 4 cores), making a couple of small changes in the code to have a fairer comparison between using %dopar% (for parallel code) and %par% (for sequential execution).

  1. Added trace = FALSE to the asreml() call, to avoid printing partial iteration details to the screen, which happened when using %do%.
  2. Used 4 pairs of sites, so I could utilize all cores in my desktop.

Execution time for the parallel version—measured with Sys.time()—was, roughly, 1/(number of cores) the time required by the sequential version.

On the (statistical) road, workshops and R

Things have been a bit quiet at Quantum Forest during the last ten days. Last Monday (Sunday for most readers) I flew to Australia to attend a couple of one-day workshops; one on spatial analysis (in Sydney) and another one on modern applications of linear mixed models (in Wollongong). This will be followed by attending The International Biometric Society Australasian Region Conference in Kiama.

I would like to comment on the workshops to look for commonalities and differences. First, both workshops heavily relied on R, supporting the idea that if you want to reach a lot of people and get them using your ideas, R is pretty much the vehicle to do so. It is almost trivial to get people to install R and RStudio before the workshop so they are ready to go. “Almost” because you have to count on someone having a bizarre software configuration or draconian security policies for their computer.

The workshop on Spatial Analysis also used WinBUGS, which with all respect to the developers, is a clunky piece of software. Calling it from R or using JAGS from R seems to me a much more sensible way of using a Bayesian approach while maintaining access to the full power of R. The workshop on linear mixed models relied on asreml-R; if you haven’t tried it, please give it a go (it is a free license for academic/research use). There were applications on multi-stage experiments, composite samples and high dimensional data (molecular information). In addition, there was an initial session on optimal design of experiments.

In my opinion, the second workshop (modern applications…) was much more successful than the first one (spatial analysis…) for a few reasons:

  • One has to limit the material to cover in a one-day workshop; if you want to cover a lot consider three days so people can digest all the material.
  • One has to avoid the “split-personality” approach to presentations; having very-basic and super-hard but nothing in the middle is not a great idea (IMHO). Pick a starting point and gradually move people from there.
  • Limit the introduction of new software. One software per day seems to be a good rule of thumb.

Something bizarre (for me, at least) was the difference between the audiences. In Sydney the crowd was a lot younger, with many trainees in biostatistics coming mostly from health research. They had little exposure to R and seemed to come from a mostly SAS shop. The crowd in Wollongong had people with a lot of experience (OK, oldish) both in statistics and R. I was expecting young people to be more conversant in R.

Tomorrow we will drive down to Kiama, sort out registration and then go to the welcome BBQ. Funny thing is that this is my first statistics conference; as I mentioned in the About page of this blog, I am a simple forester. 🙂

Surviving a binomial mixed model

A few years ago we had this really cool idea: we had to establish a trial to understand wood quality in context. Sort of following the saying “we don’t know who discovered water, but we are sure that it wasn’t a fish” (attributed to Marshall McLuhan). By now you are thinking WTF is this guy talking about? But the idea was simple; let’s put a trial that had the species we wanted to study (Pinus radiata, a gymnosperm) and an angiosperm (Eucalyptus nitens if you wish to know) to provide the contrast, as they are supposed to have vastly different types of wood. From space the trial looked like this:

The reason you can clearly see the pines but not the eucalypts is because the latter were dying like crazy over a summer drought (45% mortality in one month). And here we get to the analytical part: we will have a look only at the eucalypts where the response variable can’t get any clearer, trees were either totally dead or alive. The experiment followed a randomized complete block design, with 50 open-pollinated families in 48 blocks. The original idea was to harvest 12 blocks each year but—for obvious reasons—we canned this part of the experiment after the first year.

The following code shows the analysis in asreml-R, lme4 and MCMCglmm:

You may be wondering Where does the 3.29 in the heritability formula comes from? Well, that’s the variance of the link function that, in the case of the logit link is pi*pi/3. In the case of MCMCglmm we can estimate the degree of genetic control quite easily, remembering that we have half-siblings (open-pollinated plants):

By the way, it is good to remember that we need to back-transform the estimated effects to probabilities, with very simple code:

Even if one of your trials is trashed there is a silver lining: it is possible to have a look at survival.

Coming out of the (Bayesian) closet: multivariate version

This week I’m facing my—and many other lecturers’—least favorite part of teaching: grading exams. In a supreme act of procrastination I will continue the previous post, and the antepenultimate one, showing the code for a bivariate analysis of a randomized complete block design.

Just to recap, the results from the REML multivariate analysis (that used ASReml-R) was the following:

The corresponding MCMCglmm code is not that different from ASReml-R, after which it is modeled anyway. Following the recommendations of the MCMCglmm Course Notes (included with the package), the priors have been expanded to diagonal matrices with degree of belief equal to the number of traits. The general intercept is dropped (-1) so the trait keyword represents trait means. We are fitting unstructured (us(trait)) covariance matrices for both Block and Family, as well as an unstructured covariance matrix for the residuals. Finally, both traits follow a gaussian distribution:

Further manipulation of the posterior distributions requires having an idea of the names used to store the results. Following that, we can build an estimate of the genetic correlation between the traits (Family covariance between traits divided by the square root of the product of the Family variances). Incidentally, it wouldn’t be a bad idea to run a much longer chain for this model, so the plot of the posterior for the correlation looks better, but I’m short of time:

And that’s it! Time to congratulate Jarrod Hadfield for developing this package.