# Belief in science

It can be a touchy topic whether people who do science or make use of outcomes of science are supposed to believe in science. I can think of at least three ways that we may or may not believe in science.

# Knowledge tunnels

Imagine that we try to apply the current state of scientific knowledge to some everyday occurrence. It could be something that happens when cooking, a very particular sensation you notice in your body, anything that you have practical experience-based knowledge about, one of those particular phenomena that anyone notices when in some practical line of work. Every trade has its own specialised terminology and models of explanation that develops based on experience.

When looking closely, we will often find that the current state of scientific knowledge does not have a theory, model or explanation ready for us. This might not be because the problem is so hard to be unsolvable. It might be because the problem has simply been overlooked.

That means that if we want to maintain a world view where science has the answer, we don’t just need to believe in particular statements that science makes. We also need to trust that a scientific explanation can be developed for many cases where one doesn’t exist yet.

I can’t remember who I read who used this image (I think it might have been a mathematician): knowledge is not a solid body of work, but more like tunnels dug out of the mostly unknown. We don’t know most things, but we know some paths dug through this mass.

# Whether you believe in it or not

There is another popular saying: the good thing about it is that it works whether you believe in it or not — said by Neil DeGrasse Tyson about science and attributed to Niels Bohr about superstition.

This seems to be true, not just about deep cases such as keeping one set of methodological assumptions for your scientific work and another, say a deeply spiritual religious one, for your personal life. It seems to be true about the way we expect scientists to work and communicate on an everyday level. Scientists do not need to be committed to every statement in their writing or every hypothesis they pursue.

Dang & Bright (2021) ask what demands we should put on individual scientific contributions (e.g. journal articles). They consider assertion norms, the idea that certain types of utterances are appropriate for a certain context, and argue that scientific contributions should not be held to these norms. That is, scientific claims need not be something the researcher knows, has justification for, or believes.

They give an example of a scientist who comes up with a new hypothesis, and performs some research that supports it. Overall, however, the literature does not support it. The scientist publishes a paper advocating for the hypothesis. In a few years, further research demonstrates that it is false. They suggest that in this case, the scientist has done what we expect scientists to do, even if:

• The claims are not accurate; they are false.
• The claims are not justified, in the sense that they do not have the support of the wider literature, and the data do not conclusively support the hypothesis.
• The scientist did not fully believe the claims.

They try to explain these norms of communication with reference to how the scientific community learns. They argue that this way of communicating facilitates an intellectual division of labour: different researchers consider different ideas and theories, that might at times be at odds, and therefore there is value to allowing them to them saying conflicting things.

The rejected norms were picked as the basis of our inquiry since they had been found plausible or defensible as norms for assertion, which is at least a somewhat related activity of putting forward scientific public avowals. So why this discrepancy? In short, we think this is because the social enterprise of inquiry requires that we allow people to be more lax in certain contexts than we normally require of individuals offering testimony, and through a long process of cultural evolution the scientific community developed norms of avowal to accommodate that fact.

Another reason, pragmatically, why scientists might not be fully committed to everything they’ve written is that, at least in natural science, writing often happens by committee. And not just a committee of co-authors, but also by reviewers and editors, who also take part in negotiations about what can be claimed in any piece of peer reviewed writing. Yes, there are people who like to say that every co-author needs to be able to take full responsibility for everything written — but the real lowest common denominator seems that none of the co-authors objects too much to what is written.

# Weak citations

Finally, there appears to be two ways to think about claims when reading or writing scientific papers, that may explain that researchers can be at the same time highly critical of some claims, and somewhat credulous about other claims. Think of your typical introduction to an empirical paper, which will often have citations in passing to stylised facts that are taken to be true, but not explored at any depth — those ”neutral citations” that some think are shallow and damaging. Does that mean that scientists are lazy about the truth, just believing the first thing they see and jumping on citation bandwagons? Maybe. Because there is no other way they could write, really.

When we read or write about science, we have to divide claims into those that are currently under critical scrutiny, and those that are currently considered background. If we believe Quine, there isn’t an easy way to separate these two types of ideas, no logical division into hypothesis proper and auxiliary assumption. But we still need to make it, possibly in an arbitrary way, unless we want our research project to be an endless rabbit hole of questions. This does not mean that we have to believe the background claims in any deeper sense. They might be up for scrutiny tomorrow.

This is another reason why it’s a bad idea to try to learn a field by reading the introductions to empirical papers. Introductions are not critical expositions that synthesise evidence and give the best possible view of the state of the field. They just try to get the important background out of the way so we can move on with the work at hand.

Literature

Dang, H., & Bright, L. K. (2021). Scientific conclusions need not be accurate, justified, or believed by their authors. Synthese.

# ”Dangerous gene myths abound”

Philip Ball, who is a knowledgeable and thoughtful science writer, published an piece in the Guardian a couple of months ago about the misunderstood legacy of the human genome project: ”20 years after the human genome was first sequenced, dangerous gene myths abound”.

The human genome project published the draft reference genome for the human species 20 years ago. Ball argues, in short, that the project was oversold with promises that it couldn’t deliver, and consequently has not delivered. Instead, the genome project was good for other things that had more to do with technology development and scientific infrastructure. The sequencing of the human genome was the platform for modern genome science, but it didn’t, for example, cure cancer or uncover a complete set of instructions for building a human.

He also argues that the rhetoric of human genome hype, which did not end with the promotion of the human genome project (see the ENCODE robot punching cancer in the face, for example), is harmful. It is scientifically harmful because it oversimplifies modern genetics, and it is politically harmful because it aligns well with genetic determinism and scientific racism.

# Selling out

The breathless hype around the human genome project was embarrassing. Ball quotes some fragments, but you can to to the current human genome project site and enjoy quotes like ”it’s a transformative textbook of medicine, with insights that will give health care providers immense new powers to treat, prevent and cure disease”. This image has some metonymical truth to it — human genomics is helping medical science in different ways — but even as a metaphor, it is obviously false. You can go look at the human reference genome if you want, and you will discover that the ”text” such as it is looks more like this than a medical textbook:

TTTTTTTTCCTTTTTTTTCTTTTGAGATGGAGTCTCGCTCTGCCGCCCAGGCTGGAGTGC
AGTAGCTCGATCTCTGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCC
TCAGCCTCCTGAGTAGCTGGGACTACAGGCGCCCACCACCATGCCCAGCTAATTTTTTTT
TTTTTTTTTTTGGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTC
AATCTCCTGACCTTGTGATCCGCCCGCCTCGGCCTCCCACAGTGCTGGGATTACAGGC

This is a human Alu element from chromosome 17. It’s also in an intron of a gene, flanking a promoter, a few hundred basepairs away from an insulator (see the Ensembl genome browser) … All that is stuff that cannot be read from the sequence alone. You might be able to tell that it’s Alu if you’re an Alu genius or run a sequence recognition software, but there is no to read the other contextual genomic information, and there is no way you can tell anything about human health by reading it.

I think Ball is right that this is part of simplistic genetics that doesn’t appreciate the complexity either quantitative or molecular genetics. In short, quantitative genetics, as a framework, says that inheritance of traits between relatives is due to thousands and thousands of genetic differences each of them with tiny effects. Molecular genetics says that each of those genetic differences may operate through any of a dizzying selection of Rube Goldberg-esque molecular mechanisms, to the point where understanding one of them might be a lifetime of laboratory investigation.

Simple inheritance is essentially a fiction, or put more politely: a simple model that is useful as a step to build up a more better picture of inheritance. This is not knew; the knowledge that everything of note is complex has been around since the beginning of genetics. Even rare genetic diseases understood as monogenic are caused by sometimes thousands of different variants that happen in a particular small subset of the genome. Really simple traits, in the sense of one variant–one phenotype, seldom happen in large mixing and migrating populations like humans; they may occur in crosses constructed in the lab, or in extreme structured populations like dog breeds or possibly with balancing selection.

# Can you market thick sequencing?

Ball is also right about what it was most useful about the human genome project: it enabled research at scale into human genetic variation, and it stimulated development of sequencing methods, both generating and using DNA sequence. Lowe (2018) talks about ”thick” sequencing, a notion of sequencing that includes associated activities like assembly, annotation and distribution to a community of researchers — on top of ”thin” sequencing as determination of sequences of base pairs. Thick sequencing better captures how genome sequencing is used and stimulates other research, and aligns with how sequencing is an iterative process, where reference genomes are successively refined and updated in the face of new data, expert knowledge and quality checking.

It is hard to imagine gene editing like CRISPR being applied in any human cell without a good genome sequence to help find out what to cut out and what to put instead. It is hard to imagine the developments in functional genomics that all use short read sequencing as a read-out without having a good genome sequence to anchor the reads on. It is possible to imagine genome-wide association just based on very dense linkage maps, but it is a bit far-fetched. And so on.

Now, this raises a painful but interesting question: Would the genome project ever have gotten funded on reasonable promises and reasonable uncertainties? If not, how do we feel about the genome hype — necessary evil, unforgivable deception, something in-between? Ball seems to think that gene hype was an honest mistake and that scientists were surprised that genomes turned out to be more complicated than anticipated. Unlike him, I do not believe that most researchers honestly believed the hype — they must have known that they were overselling like crazy. They were no fools.

An example of this is the story about how many genes humans have. Ball writes:

All the same, scientists thought genes and traits could be readily matched, like those children’s puzzles in which you trace convoluted links between two sets of items. That misconception explains why most geneticists overestimated the total number of human genes by a factor of several-fold – an error typically presented now with a grinning “Oops!” rather than as a sign of a fundamental error about what genes are and how they work.

This is a complicated history. Gene number estimates are varied, but enjoy this passage from Lewontin in 1977:

The number of genes is not large

While higher organisms have enough DNA to specify from 100,000 to 1,000,000 proteins of average size, it appears that the actual number of cistrons does not exceed a few thousand. Thus, saturation lethal mapping of the fourth chromosome (Hochman, 1971) and the X chromosome (Judd, Shen and Kaufman, 1972) of Drosophila melanogbaster make it appear that there is one cistron per salivary chromosome band, of which there are 5,000 in this species. Whether 5,000 is a large or small number of total genes depends, of course, on the degree of interaction of various cistrons in influencing various traits. Nevertheless, it is apparent that either a given trait is strongly influenced by only a small number of genes, or else there is a high order of gene interactions among developmental systems. With 5,000 genes we cannot maintain a view that different parts of the organism are both independent genetically and each influenced by large number of gene loci.

I don’t know if underestimating by an few folds is worse than overestimating by a few folds (D. melanogaster has 15,000 protein-coding genes or so), but the point is that knowledgeable geneticists did not go around believing that there was a simple 1-to-1 mapping between genes and traits, or even between genes and proteins at this time. I know Lewontin is a population geneticist, and in the popular mythology population geneticists are nothing but single-minded bean counters who do not appreciate the complexity of molecular biology … but you know, they were no fools.

# The selfish cistron

One thing Ball gets wrong is evolutionary genetics, where he mixes genetic concepts that, really, have very little to do with each other despite superficially sounding similar.

Yet plenty remain happy to propagate the misleading idea that we are “gene machines” and our DNA is our “blueprint”. It is no wonder that public understanding of genetics is so blighted by notions of genetic determinism – not to mention the now ludicrous (but lucrative) idea that DNA genealogy tells you which percentage of you is “Scots”, “sub-Saharan African” or “Neanderthal”.

This passage smushes two very different parts of genetics together, that don’t belong together and have nothing to do with with the preceding questions about how many genes there are or if the DNA is a blueprint: The gene-centric view of adaptation, a way of thinking of natural selection where you imagine genetic variants (not organisms, not genomes, not populations or species) as competing for reproduction; and genetic genealogy and ancestry models, where you describe how individuals are related based on the genetic variation they carry. The gene-centric view is about adaptation, while genetic genealogy works because of effectively neutral genetics that just floats around, giving us a unique individual barcode due to the sheer combinatorics.

He doesn’t elaborate on the gene machines, but it links to a paper (Ridley 1984) on Williams’ and Dawkins’ ”selfish gene” or ”gene-centric perspective”. I’ve been on about this before, but when evolutionary geneticists say ”selfish gene”, they don’t mean ”the selfish protein-coding DNA element”; they mean something closer to ”the selfish allele”. They are not committed to any view that the genome is a blueprint, or that only protein-coding genes matter to adaptation, or that there is a 1-to-1 correspondence between genetic variants and traits.

This is the problem with correcting misconceptions in genetics: it’s easy to chide others for being confused about the parts you know well, and then make a hash of some other parts that you don’t know very well yourself. Maybe when researchers say ”gene” in a context that doesn’t sound right to you, they have a different use of the word in mind … or they’re conceptually confused fools, who knows.

Literature

Lewontin, R. C. (1977). The relevance of molecular biology to plant and animal breeding. In International Conference on Quantitative Genetics. Ames, Iowa (USA). 16-21 Aug 1976.

Lowe, J. W. (2018). Sequencing through thick and thin: Historiographical and philosophical implications. Studies in History and Philosophy of Science Part C: Studies in History and Philosophy of Biological and Biomedical Sciences, 72, 10-27.

# Using R: plyr to purrr, part 1

This is the second post about my journey towards writing more modern Tidyverse-style R code; here is the previous one. We will look at the common case of taking subset of data out of a data frame, making some complex R object from them, and then extracting summaries from those objects.

I miss the plyr package. Especially ddply, ldply, and dlply, my favourite trio of R functions of all times. Yes, the contemporary Tidyverse package dplyr is fast and neat. And those plyr functions operating on arrays were maybe overkill; I seldom used them. But plyr was so smooth, so beautiful, and — after you’ve bashed your head against it for some time and it changed your mind — so intuitive. The package still exists, but it’s retired, and we shouldn’t keep writing R code like it’s 2009, should we?

I used to like to do something like this: take a data frame, push it through a function that returns some complex object, store those objects in a list, and then map various functions over that list to extract the parts I care about. What is the equivalent in the new idiom? To do the same thing but with the purrr package, of course! purrr replaces the list-centric parts of plyr, while dplyr covers data frame-centric summarisation, mutation and so on.

For this example we will be using the lm function on subsets of data and store the model object. It’s the simple case that everyone reaches for to demonstrate these features, but it’s a bit dubious. If you find yourself fitting a lot of linear models to subsets, maybe there are other models you should think about Especially here, when the fake data just happens to come from a multilevel model with varying intercepts … But in this case, let’s simulate a simple linear regression and look at the coefficients we get out.

set.seed(20210807)

n_groups <- 10
group_sizes <- rpois(n_groups, 10)
n <- sum(group_sizes)

fake_data <- tibble(id = 1:n,
group = rep(1:n_groups,
times = group_sizes),
predictor = runif(n, 0, 1))
group_intercept <- rnorm(n_groups, 0, 1)

fake_data$response <- fake_data$predictor * 10 +
group_intercept[fake_data$group] + rnorm(n)  And here is the plyr code: First, dlply takes us from a data frame, splitting it by group, to a list of linear models. Then, ldply takes us from the list of models to a data frame of coefficients. tidy is a function from the wonderful broom package which extracts the same information as you would get in the rather unwieldy object from summary(lm), but as a data frame. library(plyr) library(broom) fit_model <- function(data) { lm(response ~ predictor, data) } models <- dlply(fake_data, "group", fit_model) result <- ldply(models, tidy)  This is what the results might looks like. Notice how ldply adds the split labels nicely into the group column, so we know which rows came from which subset.  group term estimate std.error statistic p.value 1 1 (Intercept) -0.2519167 0.5757214 -0.4375670 6.732729e-01 2 1 predictor 10.6136902 1.0051490 10.5593207 5.645878e-06 3 2 (Intercept) 3.1528489 0.6365294 4.9531864 7.878498e-04 4 2 predictor 8.2075766 1.1458702 7.1627452 5.292586e-05 5 3 (Intercept) -0.8103777 0.6901212 -1.1742542 2.786901e-01 ...  # split/map: The modern synthesis If we pull out purrr, we can get the exact same table like so. The one difference is that we get a tibble (that is, a contemporary, more well-behaved data frame) out of it instead of a base R data.frame. library(purrr) models <- map(split(fake_data, fake_data$group),
fit_model)
result <- map_df(models,
tidy,
.id = "group")

# A tibble: 80 x 6
group term        estimate std.error statistic  p.value

1 1     (Intercept)     1.67     0.773      2.16 6.32e- 2
2 1     predictor       8.67     1.36       6.39 2.12e- 4
3 2     (Intercept)     4.11     0.566      7.26 4.75e- 5
4 2     predictor       8.19     1.11       7.36 4.30e- 5
5 3     (Intercept)    -7.50     0.952     -7.89 9.99e- 5
6 3     predictor      11.5      1.75       6.60 3.03e- 4
7 4     (Intercept)   -19.8      0.540    -36.7  7.32e-13
8 4     predictor      11.5      0.896     12.8  5.90e- 8
9 5     (Intercept)   -12.4      1.03     -12.0  7.51e- 7
10 5     predictor       9.69     1.82       5.34 4.71e- 4
# … with 70 more rows


First, the base function split lets us break the data into subsets based on the values of a variable, which in this case is our group variable. The output of this function is a list of data frames, one for each group.

Second, we use map to apply a function to each element of that list. The function is the same modelling function that we used above, which shoves the data into lm. We now have our list of linear models.

Third, we apply the tidy function to each element of that list of models. Because we want the result to be one single data frame consolidating the output from each element, we use map_df, which will combine the results for us. (If we’d just use map again, we would get a list of data frames.) The .id argument tells map to add the group column that indicates what element of the list of models each row comes from. We want this to be able to identify the coefficients.

If we want to be fancy, we can express with the Tidyverse-related pipe and dot notation:

library(magrittr)

result <- fake_data %>%
coef(model)group) model_coef <- rbind(transform(lm_coef, model = "lm"), transform(lmm_coef, model = "lmm")) colnames(model_coef)[2] <- "intercept" ggplot() + geom_point(aes(x = predictor, y = response, colour = factor(group)), data = fake_data) + geom_abline(aes(slope = predictor, intercept = intercept, colour = factor(group), linetype = model), data = model_coef) + theme_bw() + theme(panel.grid = element_blank())  As advertised, the linear mixed model has the same slope in every group, and intercepts pulled closer to the mean. Now, we know that this is a good model for these fake data because we created them, and in the real world, that is obviously not the case … The point is: if we are going to fit a more complex model of the whole data, we want to be able to explore alternatives and plot them together with the data. Having elegant ways to transform data frames and summarise models at our disposal makes that process smoother. # ”It depends on what you want to do with it” Note to self: ”It depends on what you want to do with it” is the stereotypical answer to questions about method and data analysis. Ask what genetic analysis software you should use to do X, how you should filter the variants you’ve detected from sequencing, whether to mark duplicates on your aligned reads, what a genetic parameter estimate means … I just wrote a version of this answer in an email. Mea maxima culpa. Admittedly, it is probably often the technically correct answer, but it’s also useless without qualification. It always depends. Everything is everything-dependent. Goddag yxskaft. Moreover, I suspect that it often means ”I don’t know” or ”it doesn’t matter” — but you can’t say that, can you? That would sound unprofessional. Here is what to ask either the supposed expert who blurted out this non-answer, or yourself if you are about to: What are the alternatives? A couple will do to give an idea of what the field of solutions and answers is like. What does it depend on? An idea of why one want to choose one alternative over the other will do. What do we need to know? Sometimes we need preliminary data, a huge modelling effort … or just that I go read some papers before I say anything more. # Journal club of one: ”A unifying concept of animal breeding programmes” The developers of the MoBPS breeding programme simulators have published three papers about it over the last years: one about the MoBPS R package (Pook et al. 2020), one about their web server MoBPSweb (Pook et al. 2021), and one that discusses the logic of the specification for breeding programmes they use in the web interface (Simianer et al. 2021). The idea is that this specification can be used to describe breeding programmes in a precise and interoperable manner. The latter — about breeding programme specification — reads as if the authors had a jolly good time thinking about what breeding and breeding programmes are; at least, I had such feelings while reading it. It is accompanied by an editorial by Simianer (2021) talking about what he thinks are the important research directions for animal breeding research. Using simulation to aid breeding programme design is one of them. # Defining and specifying breeding programmes After defining a breeding programme in a narrow genetic sense as a process achieving genetic change (more about that later), Simianer et al. (2021) go on to define a specification of such a breeding programme — or more precisely, of a model of a breeding programme. They use the word ”definition” for both things, but they really talk about two different things: defining what ”a breeding programme” is and specifying a particular model of a particular breeding programme. Here, I think it is helpful to think of Guest & Martin’s (2020) distinction, borrowed from psychology, between a specification of a model and an implementation of a model. A specification is a description of a model based in natural language or math. Breeding programme modelling often takes the shape of software packages, where the software implementation is the model and a specification is quite vague. Simianer et al. (2021) can be seen as a step towards a way of writing specifications, on a higher level in Guest & Martin’s hierarchy, of what such a breeding programme model should achieve. They argue that such a specification (”formal description”) needs to be comprehensive, unambiguous and reproducible. They claim that this can be achieved with two parts: the breeding environment and the breeding structure. The breeding environment includes: • founder populations, • quantitative genetic parameters for traits, • genetic architectures for traits, • economic values for traits in breeding goal, • description of genomic information, • description of breeding value estimation methods. Thus, the ”formal” specification depends on a lot of information that is either unknowable in practice (genetic architecture), estimated with error (genetic parameters), hard to describe other than qualitatively (founder population) and dependent on particular software implementations and procedures (breeding value estimation). This illustrates the need to distinguish the map from the territory — to the extent that the specification is exact, it describes a model of a breeding programme, not a real breeding programme. The other part of their specification is the graph-based model of breeding structure. I think this is their key new idea. The breeding structure, in their specification, consists of nodes that represent groups of elementary objects (I would say ”populations”) and edges that represent transformations that create new populations (such as selection or mating) or are a shorthand for repeating groups of edges and nodes. The elementary objects could be individuals, but they also give the example of gametes and genes (I presume they mean in the sense of alleles) as possible elementary objects. One could also imagine groups of genetically identical individuals (”genotypes” in a plant breeding sense). Nodes contain a given number of individuals, and can also have a duration. Edges are directed, and correspond to processes such as ageing, selection, reproduction, splitting or merging populations. They will carry attributes related to the transformation. Edges can have a time associated with them that it takes for the transformation to happen (e.g. for animals to gestate or grow to a particular age). Here is an example from Pook et al. (2021) of a breeding structure graph for dairy cattle: If we ignore the red edges for now, we can follow the flow of reproduction (yellow edges) and selection (green edges): The part on the left is what is going on in the breeding company: cows (BC-Cows) reproduce with selected bulls (BC-SelectedBulls), and their offspring become the next generation of breeding company cows and bulls (BC-NextCows and BC-NextBulls). On the right is the operation of a farm, where semen from the breeding company is used to inseminate cows and heifers (heifer, cow-L1, cow-L2, cow-L3) to produce calfs (calf-h, calf-L1, calf-L2, calf-L3). Each cycle each of these groups, through a selection operation, give rise to the next group (heifers becoming cows, first lactation cows becoming second lactation cows etc). # Breeding loops vs breeding graphs vs breeding forms Except for the edges that specify breeding operations, there is also a special meta-edge type, the repeat edge, that is used to simplify breeding graphs with repeated operations. A useful edge class to describe breeding programmes that are composed of several breeding cycles is ”repeat.” It can be used to copy resulting nodes from one breeding cycle into the nodes of origin of the next cycle, assuming that exactly the same breeding activities are to be repeated in each cycle. The “repeat” edge has the attribute “number of repeats” which allows to determine the desired number of cycles. In the MoBPSweb paper (Pook et al. 2020), they describe how it is implemented in MoBPS: Given a breeding specification in MoBPSweb JSON format, the simulator will generate a directed graph by copying the nodes on the breeding cycle as many times as is specified by the repeat number. In this way, repeat edges are eliminated to make the breeding graph acyclic. The conversion of the breeding scheme itself is done by first detecting if the breeding scheme has any “Repeat” edges (Simianer et al. 2020), which are used to indicate that a given part of the breeding programme is carried out multiple times (breeding cycles). If that is the case, it will subsequently check which nodes can be generated without the use of any repeat. Next, all repeats that can be executed based on the already available nodes are executed by generating copies of all nodes between the node of origin and the target node of the repeat (including the node of origin and excluding the target node). Nodes generated via repeat are serial-numbered via “_1,” “_2” etc. to indicate the repeat number. This procedure is repeated until all repeat edges are resolved, leading to a breeding programme without any repeats remaining. There are at least three ways to specify the breeding structures for breeding programme simulations: these breeding graphs, breeding loops (or more generally, specifying breeding in a programming language) and breeding forms (when you get a pre-defined breeding structure and are allowed fill in the numbers). If I’m going to compare the graph specification to what I’m more familiar with, this is how you would create a breeding structure in AlphaSimR: library(AlphaSimR) ## Breeding environment founderpop <- runMacs(nInd = 100, nChr = 20) simparam <- SimParamnew(founderpop)
simparam$setSexes("yes_sys") simparam$addTraitA(nQtlPerChr = 100)
simparam$setVarE(h2 = 0.3) ## Breeding structure n_time_steps <- 10 populations <- vector(mode = "list", length = n_time_steps + 1) populations[[1]] <- newPop(founderpop, simParam = simparam) for (gen_ix in 2:(n_time_steps + 1)) { ## Breeding cycle happens here }  In the AlphaSimR script, the action typically happens within the loop. You apply different functions on population objects to make your selection, move individuals between parts of the breeding programme, create offspring etc. That is, in the MoBPS breeding structure, populations are nodes and actions are edges. In AlphaSimR, populations are objects and operations are functions. In order to not have to copy paste your breeding code, you use the control flow structures of R to make a loop (or some functional equivalent). In MoBPS graph structure, in order to not have to create every node and edge manually, you use the Repeat edge. Breeding graphs with many repeat edges with different times attached to them have the potential to be complicated, and the same is true of breeding loops. I would have to use both of them more to have an opinion about what is more or less intuitive. Now that we’ve described what they do in the paper, let’s look at some complications. # Formal specifications only apply to idealised breeding programmes The authors claim that their concept provides a formal breeding programme specification (in their words, ”formal description”) that can be fully understood and implemented by breeders. It seems like the specification fails to live up to this ambition, and it appears doubtful whether any type of specification can. This is because they do not distinguish between specifying a model of a breeding programme and specifying a real implementation of a breeding programme. First, as mentioned above, the ”breeding environment” as described by them, contains information that can never be specified for any real population, such as the genetic architecture of complex traits. Second, their breeding structure is described in terms of fixed numbers, which will never be precise due to mortality, conception rates, logistics and practical concerns. They note such fluctuations in population size as a limitation in the Discussion. To some extent, random mortality, reproductive success etc an be modelled by putting random distributions on various parameters. (I am not sure how easy this is to do in the MoBPS framework; it is certainly possible.) However, this adds uncertainty about what these hyperparameter should be and whether they are realistic. Such complications would just be nit-picking if the authors had not suggested that their specification can be used to communicate breeding programmes between breeders and between breeders and authorities, such as when a breeding programme is seeking approval. They acknowledge that the authorities, for example in the EU, want more detailed information that are beyond the scope of their specification. # And the concept is not very formal in the first place Despite the claimed formality, every class of object in the breeding structure is left open, with many possible actions and many possible attributes that are never fully defined. It is somewhat ambiguous what is to be the ”formal” specification — it cannot be the description in the paper as it is not very formal or complete; it shouldn’t be the implementation in MoBPS and MoBPSweb, as the concept is claimed to be universal; maybe it is the JSON specification of the breeding structure and background as described in the MoBPSweb paper (Pook et al. 2020). The latter seems the best candidate for a well-defined formal way to specify breeding programme models, but then again, the JSON format appears not to have a published specification, and appears to contain implementation-specific details relating to MoBPS. This also matters to the suggested use of the specification to communicate real breeding programme designs. What, precisely, is it that will be communicated? Are breed societies and authorities expected to communicate with breeding graphs, JSON files, or with verbal descriptions using their terms (e.g. ”breeding environment”, ”breeding structure”, their node names and parameters for breeding activities)? # There is almost never a need for a definition As I mentioned before, the paper starts by asking what a breeding programme is. They refer to different descriptions of breeding programme design from textbooks, and a legal definition from EU regulation 2016/1012; article 2, paragraph 26, which goes: ‘breeding programme’ means a set of systematic actions, including recording, selection, breeding and exchange of breeding animals and their germinal products, designed and implemented to preserve or enhance desired phenotypic and/or genotypic characteristics in the target breeding population. There seems to be some agreement that a breeding programme, in addition to being the management of reproduction of a domestic animal population, also is systematic and goal-directed activity. Despite these descriptions of breeding programmes and their shared similarity, the authors argue that there is no clear formal definition of what a breeding programme is, and that this would be useful to delineate and specify breeding programmes. They define a breeding programme as an organised process that aims to change the genetic composition in a desired direction, from one group of individuals to a group of individuals at a later time. The breeding programme comprises those individuals and activities that contribute to this process. For example, crossbred individuals in a multiplier part of a terminal crossbreeding programme would be included to the extent that they contribute information to the breeding of nucleus animals. We define a breeding programme as a structured, man-driven process in time that starts with a group of individuals X at time $t_1$ and leads to a group of individuals Y at time $t_2 > t_1$. The objective of a breeding programme is to transform the genetic characteristics of group X to group Y in a desired direction, and a breeding programme is characterized by the fact that the implemented actions aim at achieving this transformation. They actually do not elaborate on what it means that a genetic change has direction, but since they want the definition to apply both to farm animal and conservation breeding programmes, the genetic goals could be formulated both in terms of changes in genetic values for traits and in terms of genetic relationships. Under many circumstances, this is a reasonable proxy also for an economic target: The breeding structures and interventions considered in theoretical breeding programme designs can often be evaluated in terms of their effect on the response to selection, and if the response improves, so will the economic benefit. However, this definition seems a little unnecessary and narrow. If you wanted to, say, add a terminal crossbreeding step to the simulation and evaluate the performance in terms of the total profitability of the crossbreeding programme (that is, something that is outside of the breeding programme in the sense of the above definition), nothing is stopping you, and the idea is not in principle outside of the scope of animal breeding. # Finally, an interesting remark about efficiency When discussing the possibility of using their concept to communicate breeding programmes to authorities when seeking approval the authors argue that authorities should not take efficiency of the breeding programme into account when they evaluate breeding programmes for approval. They state this point forcefully without explaining their reasoning: It should be noted, though, that an evaluation of the efficiency of breeding programme is not, and should never be, a precondition for the approval of a breeding programme by the authorities. This raises the question: why not? There might be both environmental, economical and animal ethical reasons to consider not approving breeding programmes that can be shown to make inefficient use of resources. Maybe such evaluation would be impractical — breeding programme analysis and simulation might have to be put on a firmer scientific grounding and be made more reproducible and transparent before we trust it to make such decisions — but efficiency does seem like an appropriate thing to want in a breeding scheme, also from the perspective of society and the authorities. I am not advocating any particular new regulation for breeding programmes here, but I wonder where the ”should never” came from. This reads like a comment added to appease a reviewer — the passage is missing from the preprint version. Literature Pook, T., Schlather, M., & Simianer, H. (2020a). MoBPS-modular breeding program simulator. G3: Genes, Genomes, Genetics, 10(6), 1915-1918. https://academic.oup.com/g3journal/article/10/6/1915/6026363 Pook, T., Büttgen, L., Ganesan, A., Ha, N. T., & Simianer, H. (2021). MoBPSweb: A web-based framework to simulate and compare breeding programs. G3, 11(2), jkab023. https://academic.oup.com/g3journal/article/11/2/jkab023/6128572 Simianer, H., Büttgen, L., Ganesan, A., Ha, N. T., & Pook, T. (2021). A unifying concept of animal breeding programmes. Journal of Animal Breeding and Genetics, 138 (2), 137-150. https://onlinelibrary.wiley.com/doi/full/10.1111/jbg.12534 Simianer, H. (2021), Harvest Moon: Some personal thoughts on past and future directions in animal breeding research. J Anim Breed Genet, 138: 135-136. https://doi.org/10.1111/jbg.12538 Guest, O., & Martin, A. E. (2020). How computational modeling can force theory building in psychological science. Perspectives on Psychological Science, 1745691620970585. https://journals.sagepub.com/doi/full/10.1177/1745691620970585 # Paper: ”Genetic variation in recombination rate in the pig” Our paper on genetic variation in recombination in the pig just came out the other week. I posted about it already when it was a preprint, but we dug a little deeper into some of the results in response to peer review, so let us have a look at it again. Summary Recombination between chromosomes during meiosis leads to shuffling of genetic material between chromosomes, creating new combinations of alleles. Recombination rate varies, though, between parts of the genome, between sexes, and between individuals. Illustrating that, here is a figure from the paper showing how recombination rate varies along the chromosomes of the pig genome. Female recombination rate is higher than male recombination rate on most chromosomes, and in particular in regions of higher recombination rate in the middle of certain chromosomes. Fig 2 from the paper: average recombination rate in 1 Mbp windows along the pig genome. Each line is a population, coloured by sexes. In several other vertebrates, part of that individual variation in recombination rate (in the gametes passed on by that individual) is genetic, and associated with regions close to known meiosis-genes. It turns out that this is the case in the pig too. In this paper, we estimated recombination rates in nine genotyped pedigree populations of pigs, and used that to perform genome-wide association studies of recombination rate. The heritability of autosomal recombination rate was around 0.07 in females, 0.05 in males. The major genome-wide association hit on chromosome 8, well known from other mammals, overlapped the RNF212 gene in most populations in females, and to a lesser extent in males. Fig 6 from the paper, showing genome-wide association results for eight of the populations (one had too few individuals with recombination rate estimates after filtering for GWAS). The x-axis is position in the genome, and the y-axis is the negative logarithm of the p-value from a linear mixed model with repeated measures. One of the things we added after the preprint is a meta-analysis of genome-wide association over all the populations (separated by sex). In total, there were six associated regions, five of which are close to known recombination genes: RNF212, SHOC1, SYCP2, MSH4 and HFM1. In particular, several of the candidates are genes involved in whether a double strand break resolves as a crossover or non-crossover. However, we do not have the genomic resolution to know whether these are actually the causative genes; there are significant markers overlapping many genes, and the candidate genes are not always the closest gene. How well does the recombination landscape agree with previous maps? The recombination landscapes accord pretty well with Tortereau et al.’s (2012) maps. We find a similar sex difference, with higher recombination in females on all autosomes except chromosome 1 and 13, and a stronger association with GC content in females. However, our recombination rates tend to be higher, possibly due to some overestimation. Different populations, where recombinations are estimated independently, also have similar recombination landscapes. But it doesn’t agree so well with Lozada-Soto et al. (2021), does it? No, that is true. Between preprint and finished version, Lozada-Soto et al. (2021) published a genome-wide association study of recombination rate in the pig. They found heritabilities of recombination rate of a similar magnitude as we did, but their genome-wide association results are completely different. We did not find the hits that they found, and they did not find the hits we found, or any previously known candidate genes for recombination. To be honest, I don’t have a good explanation for these differences. How about recombination hotspots and PRDM9? At a very fine scale, most recombination tends to occur in hotspots of around a few kilobasepairs. As this study used pedigrees and SNP chips with much coarser density than this, we cannot say much about the fine-scale recombination landscape. We work, at the finest, with windows of 1 Mbp. However, as the pig appears to have a working and rapidly evolving PRDM9 gene (encoding a protein that is responsible for recombination hotspot targeting), the pig probably has a PRDM9-based landscape of hotspots just like humans and mice (Baker et al. 2019). Tortereau et al. (2012) found a positive correlation between counts of the PRDM9 DNA-binding motif and recombination rate, which is biologically plausible, as more PRDM9 motifs should mean more hotspots. So, we estimated this correlation for comparison, but found only a very weak relationship — this is one point where our results are inconsistent with previous maps. This might be because of changes in improved pig genome assembly we use, or it might be an indication that we have worse genomic resolution due to the genotype imputation involved in our estimation. However, one probably shouldn’t expect to find strong relationships between a process at the kilobasepair-scale when using windows of 1 Mbp in the first place. Can one breed for increased recombination to improve genetic gain? Not really. Because recombination breaks up linkage disequlibrium between causative variants, higher recombination rate could reveal genetic variation for selection and improve genetic gain. However, previous studies suggest that recombination rate needs to increase quite a lot (two-fold or more) to substantially improve breeding (Battagin et al. 2016). We made some back of the envelope quantitative genetic calculations on the response to selection for recombination, and it would be much smaller than that. Literature Johnsson M*, Whalen A*, Ros-Freixedes R, Gorjanc G, Chen C-Y, Herring WO, de Koning D-J, Hickey JM. (2021) Genetics variation in recombination rate in the pig. Genetics Selection Evolution (* equal contribution) Tortereau, F., Servin, B., Frantz, L., Megens, H. J., Milan, D., Rohrer, G., … & Groenen, M. A. (2012). A high density recombination map of the pig reveals a correlation between sex-specific recombination and GC content. BMC genomics, 13(1), 1-12. Lozada‐Soto, E. A., Maltecca, C., Wackel, H., Flowers, W., Gray, K., He, Y., … & Tiezzi, F. (2021). Evidence for recombination variability in purebred swine populations. Journal of Animal Breeding and Genetics, 138(2), 259-273. Baker, Z., Schumer, M., Haba, Y., Bashkirova, L., Holland, C., Rosenthal, G. G., & Przeworski, M. (2017). Repeated losses of PRDM9-directed recombination despite the conservation of PRDM9 across vertebrates. Elife, 6, e24133. Battagin, M., Gorjanc, G., Faux, A. M., Johnston, S. E., & Hickey, J. M. (2016). Effect of manipulating recombination rates on response to selection in livestock breeding programs. Genetics Selection Evolution, 48(1), 1-12. # Preprint: ”Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems” We have a new preprint posted to Biorxiv looking into the genetic basis of bone strength and other bone properties in crossbred laying hens in two different housing environments (furnished cages and floor pens). Here are the citation and abstract: Martin Johnsson, Helena Wall, Fernando A Lopes Pinto, Robert H. Fleming, Heather A. McCormack, Cristina Benavides-Reyes, Nazaret Dominguez-Gasca, Estefania Sanchez-Rodriguez, Ian C. Dunn, Alejandro B. Rodriguez-Navarro, Andreas Kindmark, Dirk-Jan de Koning (2021) Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems. bioRxiv 2021.06.21.449243 Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density and bone composition, as well as body weight, in 860 commercial crossbred laying hens from two different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds, and performed a genome-wide association study of bone properties and body weight. As expected, the two housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralisation and chemical composition. Genome-scans either combining or independently analysing the two housing systems revealed no genome-wide significant loci for bone breaking strength. We detected three loci for body weight that were shared between the housing systems on chromosomes 4, 6 and 27 (either genome-wide significant or suggestive when the housing systems were analysed individually) and these coincide with associations for bone length. In summary, we found substantial differences in bone strength, content and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes support gene-by-environment interactions with housing system. The background is that bone quality is a serious problem for laying hens; that housing systems that allow for more movement are known to lead to stronger bones; and that previous work on the genetics of bone parameters comes mostly from pure lines or from experimental intercrosses between divergent lines. Here, we study commercial crossbred laying hens from two different companies. Being housed in a floor pen, where there is more opportunity for physical activity, or in a furnished cage makes a big difference to bone breaking strength. For comparison, we also show body weight, which is not much different between the housing environments. This difference was accompanied by differences in bone composition (see details in the paper). And here are the Manhattan plots from genome-wide association: bone strength shows no major loci, as opposed to body weight, which has strong associations that are shared between the housing systems. And if we compare the genome-wide associations, marker for marker, between the housing systems, there is nothing in common between the suggestive associations for bone strength. (Body weight below for comparison.) This includes not detecting major loci for bone strength that have been found in pure lines of chickens. We think this is due to gene-by-environment interactions with housing (i.e. physical activity). This might be a complication for genomic selection for bone quality, as selection might need to be targeted to different housing systems. Finally, the three strong association for body weight shown above overlap previously detected loci on chromosomes 4, 6, and 27. We do not have the genomic resolution to nominate candidate genes with any confidence, but the chromosome 4 locus overlaps both the CCKAR gene, which is a strong candidate for growth and body mass in the chicken and the LCORL/NCAPG locus, which has been associated with body size in several species. These regions (plus a fourth one) are also associated with bone length: # Convincing myself about the Monty Hall problem Like many others, I’ve never felt that the solution to the Monty Hall problem was intuitive, despite the fact that explanations of the correct solution are everywhere. I am not alone. Famously, columnist Marilyn vos Savant got droves of mail from people trying to school her after she had published the correct solution. The problem goes like this: You are a contestant on a game show (based on a real game show hosted by Monty Hall, hence the name). The host presents you with three doors, one of which contains a prize — say, a goat — and the others are empty. After you’ve made your choice, the host opens one of the doors, showing that it is empty. You are now asked whether you would like to stick to your initial choice, or switch to the other door. The right thing to do is to switch, which gives you 2/3 probability of winning the goat. This can be demonstrated in a few different ways. A goat is a great prize. Image: Casey Goat by Pete Markham (CC BY-SA 2.0) So I sat down to do 20 physical Monty Hall simulations on paper. I shuffled three cards with the options, picked one, and then, playing the role of the host, took away one losing option, and noted down if switching or holding on to the first choice would have been the right thing to do. The results came out 13 out of 20 (65%) wins for the switching strategy, and 7 out of 20 (35%) for the holding strategy. Of course, the Monty Hall Truthers out there must question whether this demonstration in fact happened — it’s too perfect, isn’t it? The outcome of the simulation is less important than the feeling that came over me as I was running it, though. As I was taking on the role of the host and preparing to take away one of the losing options, it started feeling self-evident that the important thing is whether the first choice is right. If the first choice is right, holding is the right strategy. If the first choice is wrong, switching is the right option. And the first choice, clearly, is only right 1/3 of the time. In this case, it was helpful to take the game show host’s perspective. Selvin (1975) discussed the solution to the problem in The American Statistician, and included a quote from Monty Hall himself: Monty Hall wrote and expressed that he was not ”a student of statistics problems” but ”the big hole in your argument is that once the first box is seen to be empty, the contestant cannot exchange his box.” He continues to say, ”Oh, and incidentally, after one [box] is seen to be empty, his chances are no longer 50/50 but remain what they were in the first place, one out of three. It just seems to the contestant that one box having been eliminated, he stands a better chance. Not so.” I could not have said it better myself. # A generalised problem Now, imagine the same problem with a number d number of doors, w number of prizes and o number of losing doors that are opened after the first choice is made. We assume that the losing doors are opened at random, and that switching entails picking one of the remaining doors at random. What is the probability of winning with the switching strategy? The probability of picking the a door with or without a prize is: $\Pr(\text{pick right first}) = \frac{w}{d}$ $\Pr(\text{pick wrong first}) = 1 - \frac{w}{d}$ If we picked a right door first, we have w – 1 winning options left out of d – o – 1 doors after the host opens o doors: $\Pr(\text{win\textbar right first}) = \frac{w - 1}{d - o - 1}$ If we picked the wrong door first, we have all the winning options left: $\Pr(\text{win\textbar wrong first}) = \frac{w}{d - o - 1}$ Putting it all together: $\Pr(\text{win\textbar switch}) = \Pr(\text{pick right first}) \cdot \Pr(\text{win\textbar right first}) + \\ + \Pr(\text{pick wrong first}) \cdot \Pr(\text{win\textbar wrong first}) = \\ = \frac{w}{d} \frac{w - 1}{d - o - 1} + (1 - \frac{w}{d}) \frac{w}{d - o - 1}$ As before, for the hold strategy, the probability of winning is the probability of getting it right the first time: $\Pr(\text{win\textbar hold}) = \frac{w}{d}$ With the original Monty Hall problem, w = 1, d = 3 and o = 1, and therefore $\Pr(\text{win\textbar switch}) = \frac{1}{3} \cdot 0 + \frac{2}{3} \cdot 1$ Selvin (1975) also present a generalisation due to Ferguson, where there are n options and p doors that are opened after the choice. That is, w = 1, d = 3 and o = 1. Therefore, $\Pr(\text{win\textbar switch}) = \frac{1}{n} \cdot 0 + (1 - \frac{1}{n}) \frac{1}{n - p - 1} = \frac{n - 1}{n(n - p - 1)}$ which is Ferguson’s formula. Finally, in Marilyn vos Savant’s column, she used this thought experiment to illustrate why switching is the right thing to do: Here’s a good way to visualize what happened. Suppose there are a million doors, and you pick door #1. Then the host, who knows what’s behind the doors and will always avoid the one with the prize, opens them all except door #777,777. You’d switch to that door pretty fast, wouldn’t you? That is, w = 1 still, d = 106 and o = 106 – 2. $\Pr(\text{win\textbar switch}) = 1 - \frac{1}{10^6}$ It turns out that the solution to the generalised problem is that it is always better to switch, as long as there is a prize, and as long as the host opens any doors. One can also generalise it to choosing sets of more than one door. This makes some intuitive sense: as long as the host takes opens some doors, taking away losing options, switching should enrich for prizes. # Some code To be frank, I’m not sure I have convinced myself of the solution to the generalised problem yet. However, using the code below, I did try the calculation for all combinations of total number of doors, prizes and doors opened up to 100, and in all cases, switching wins. That inspires some confidence, should I end up on a small ruminant game show. The code below first defines a wrapper around R’s sampling function, which has a very annoying alternative behaviour when fed a vector of length one, to be able to build a computational version of my physical simulation. Finally, we have a function for the above formulae. (See whole thing on GitHub if you are interested.) ## Wrap sample into a function that avoids the "convenience" ## behaviour that happens when the length of x is one sample_safer <- function(to_sample, n) { assert_that(n <= length(to_sample)) if (length(to_sample) == 1) return(to_sample) else { return(sample(to_sample, n)) } } ## Simulate a generalised Monty Hall situation with ## w prizes, d doors and o doors that are opened. sim_choice <- function(w, d, o) { ## There has to be less prizes than unopened doors assert_that(w < d - o) wins <- rep(1, w) losses <- rep(0, d - w) doors <- c(wins, losses) ## Pick a door choice <- sample_safer(1:d, 1) ## Doors that can be opened to_open_from <- which(doors == 0) ## Chosen door can't be opened to_open_from <- to_open_from[to_open_from != choice] ## Doors to open to_open <- sample_safer(to_open_from, o) ## Switch to one of the remaining doors possible_switches <- setdiff(1:d, c(to_open, choice)) choice_after_switch <- sample_safer(possible_switches , 1) result_hold <- doors[choice] result_switch <- doors[choice_after_switch] c(result_hold, result_switch) } ## Formulas for probabilities mh_formula <- function(w, d, o) { ## There has to be less prizes than unopened doors assert_that(w < d - o) p_win_switch <- w/d * (w - 1)/(d - o - 1) + (1 - w/d) * w / (d - o - 1) p_win_hold <- w/d c(p_win_hold, p_win_switch) } ## Standard Monty Hall mh <- replicate(1000, sim_choice(1, 3, 1))  > mh_formula(1, 3, 1) [1] 0.3333333 0.6666667 > rowSums(mh)/ncol(mh) [1] 0.347 0.653 # The Monty Hall problem problem Guest & Martin (2020) use this simple problem as their illustration for computational model building: two 12 inch pizzas for the same price as one 18 inch pizza is not a good deal, because the 18 inch pizza contains more food. Apparently this is counter-intuitive to many people who have intuitions about inches and pizzas. They call the risk of having inconsistencies in our scientific understanding because we cannot intuitively grasp the implications of our models ”The pizza problem”, arguing that it can be ameliorated by computational modelling, which forces you to spell out implicit assumptions and also makes you actually run the numbers. Having a formal model of areas of circles doesn’t help much, unless you plug in the numbers. The Monty Hall problem problem is the pizza problem with a vengeance; not only is it hard to intuitively grasp what is going on in the problem, but even when presented with compelling evidence, the mental resistance might still remain and lead people to write angry letters and tweets. Literature Guest, O, & Martin, AE (2020). How computational modeling can force theory building in psychological science. Preprint. Selvin, S (1975) On the Monty Hall problem. The American Statistician 29:3 p.134. # Showing a difference in mean between two groups, take 2 A couple of years ago, I wrote about the paradoxical difficulty of visualising a difference in means between two groups, while showing both the data and some uncertainty interval. I still feel like many ills in science come from our inability to interpret a simple comparison of means. Anything with more than two groups or a predictor that isn’t categorical makes things worse, of course. It doesn’t take much to overwhelm the intuition. My suggestion at the time was something like this — either a panel that shows the data an another panel with coefficients and uncertainty intervals; or a plot that shows the with lines that represent the magnitude of the difference at the upper and lower limit of the uncertainty interval. Alternative 1 (left), with separate panels for data and coefficient estimates, and alternative 2 (right), with confidence limits for the difference shown as vertical lines. For details, see the old post about these graphs. Here is the fake data and linear model we will plot. If you want to follow along, the whole code is on GitHub. Group 0 should have a mean of 4, and the difference between groups should be two, and as the graphs above show, our linear model is not too far off from these numbers. library(broom) data <- data.frame(group = rep(0:1, 20)) data$response <- 4 + data$group * 2 + rnorm(20) model <- lm(response ~ factor(group), data = data) result <- tidy(model)  Since the last post, a colleague has told me about the Gardner-Altman plot. In a paper arguing that confidence intervals should be used to show the main result of studies, rather than p-values, Gardner & Altman (1986) introduced plots for simultaneously showing confidence intervals and data. Their Figure 1 shows (hypothetical) systolic blood pressure data for a group of diabetic and non-diabetic people. The left panel is a dot plot for each group. The right panel is a one-dimensional plot (with a different scale than the right panel; zero is centred on the mean of one of the groups), showing the difference between the groups and a confidence interval as a point with error bars. There are functions for making this kind of plot (and several more complex alternatives for paired comparisons and analyses of variance) in the R package dabestr from Ho et al. (2019). An example with our fake data looks like this: Alternative 3: Gardner-Altman plot with bootstrap confidence interval. library(dabestr) bootstrap <- dabest(data, group, response, idx = c("0", "1"), paired = FALSE) bootstrap_diff <- mean_diff(bootstrap) plot(bootstrap_diff)  While this plot is neat, I think it is a little too busy — I’m not sure that the double horizontal lines between the panels or the shaded density for the bootstrap confidence interval add much. I’d also like to use other inference methods than bootstrapping. I like how the scale of the right panel has the same unit as the left panel, but is offset so the zero is at the mean of one of the groups. Here is my attempt at making a minimalistic version: Alternative 4: Simplified Garner-Altman plot. This piece of code first makes the left panel of data using ggbeeswarm (which I think looks nicer than the jittering I used in the first two alternatives), then the right panel with the estimate and approximate confidence intervals of plus/minus two standard errors of the mean), adjusts the scale, and combines the panels with patchwork. library(ggbeeswarm) library(ggplot2 library(patchwork) ymin <- min(data$response)
ymax <- max(data\$response)

plot_points_ga <- ggplot() +
geom_quasirandom(aes(x = factor(group), y = response),
data = data) +
xlab("Group") +
ylab("Response") +
theme_bw() +
scale_y_continuous(limits = c(ymin, ymax))

height_of_plot <- ymax-ymin

group0_fraction <- (coef(model)[1] - ymin)/height_of_plot

diff_min <- - height_of_plot * group0_fraction

diff_max <- (1 - group0_fraction) * height_of_plot

plot_difference_ga <- ggplot() +
geom_pointrange(aes(x = term, y = estimate,
ymin = estimate - 2 * std.error,
ymax = estimate + 2 * std.error),
data = result[2,]) +
scale_y_continuous(limits = c(diff_min, diff_max)) +
ylab("Difference") +
xlab("Comparison") +
theme_bw()

(plot_points_ga | plot_difference_ga) +
plot_layout(widths = c(0.75, 0.25))


Literature

Gardner, M. J., & Altman, D. G. (1986). Confidence intervals rather than P values: estimation rather than hypothesis testing. British Medical Journal

Ho, J., Tumkaya, T., Aryal, S., Choi, H., & Claridge-Chang, A. (2019). Moving beyond P values: data analysis with estimation graphics. Nature methods.

# Journal club of one: ”Genome-wide enhancer maps link risk variants to disease genes”

(Here is a a paper from a recent journal club.)

Nasser et al. (2021) present a way to prioritise potential noncoding causative variants. This is part of solving the fine mapping problem, i.e. how to find the underlying causative variants from genetic mapping studies. They do it by connecting regulatory elements to genes and overlapping those regulatory elements with variants from statistical fine-mapping. Intuitively, it might not seem like connecting regulatory elements to genes should be that hard, but it is a tricky problem. Enhancers — because that is the regulatory element most of this is about; silencers and insulators get less attention — do not carry any sequence that tells us what gene they will act on. Instead, this needs to be measured or predicted somehow. These authors go the measurement route, combining chromatin sequencing with chromosome conformation capture.

This figure from the supplementary materials show what the method is about:

Additional figure 1 from Nasser et al. (2021) showing an overview of the workflow and an example of two sets of candidate variants derived from-fine mapping, each with variants that overlap enhancers connected to IL10.

They use chromatin sequence data (such as ATAC-seq, histone ChIP-seq or DNAse-seq) in combination with HiC chromosome conformation capture data to identify enhancers and connect them to genes (this was developed earlier in Fulco et al. 2019). The ”activity-by-contact model” means to multiply the contact frequency (from HiC) between promoter and enhancer with the enhancer activity (read counts from chromatin sequencing), standardised by the total contact–activity product with all elements within a window. Fulco et al. (2019) previously found that this conceptually simple model did well at connecting enhancers to genes (as evaluated with a CRISPR inhibition assay called CRISPRi-FlowFISH, which we’re not going into now, but it’s pretty ingenious).

In order to use this for fine-mapping, they calculated activity-by-contact maps for every gene combined with every open chromatin element within 5 Mbp for 131 samples from ENCODE and other sources. The HiC data were averaged of contacts in ten cell types, transformed to be follow a power-law distribution. That is, they did not do the HiC analysis per cell type, but use the same average HiC contact matrix combined with chromatin data from different cell types. Thus, the specificity comes from the promoters and enhancers that are called as active in each sample — I assume this is because the HiC data comes from a different (and smaller) set of cell types than the chromatin sequencing. Element–gene pairs that reached above a threshold were considered connected, for a total of about six million connections, involving 23,000 genes and 270,000 enhancers. On average, a gene had 2.8 enhancers and an enhancer connected to 2.7 genes.

They picked putative causative variants by overlapping the variant sets with these activity-by-contact maps and selecting the highest scoring enhancer gene pair.They used fine-mapping results from multiple previous studies. These variants were estimated with different methods, but they are all some flavour of fine-mapping by variable selection. Statistical fine mapping estimate sets of variants, called credible sets, that have high posterior probability of being the causative variant. They included only completely noncoding credible sets, i.e. those that did not include a coding sequence or splice variant. They applied this to 72 traits in humans, generating predictions for ~ 5000 noncoding credible sets of variants.

# Did it work?

Variants for fine-mapping were enriched in connected enhancers more than in open chromatin in general, in cell types that are relevant to the traits. In particular, inflammatory bowel disease variants were enriched in enhancers in 65 samples, including immune cell types and gut cells. The strongest enrichment was in activated dendritic cells.

They used a set of genes previously known to be involved in inflammatory bowel disease, assuming that they were the true causative genes for their respective noncoding credible sets, and then compared their activity-by-contact based prioritisation of the causative gene to simply picking the closest gene. Picking the closest gene was right in 30 out of 37 sets. Picking the gene with the highest activity-by-contact score was right in 17 cases out of 18 sets that overlapped an activity-by-contact enhancer. Thus, this method had higher precision but worse recall. They also tested a number of eQTL-based, enrichment and enhancer–gene mapping methods, that did worse.

# What it tells us about causative variants

Most of the putative causative variants, picked based on maximising activity-by-contact, were close to the proposed gene (median 13 kbp) and most involved the closest gene (77%). They were often found their putative causative variants to be located in enhancers that were only active in a few cell- or tissue types (median 4), compared to the promoters of the target genes, that were active in a broader set (median 120). For example, in inflammatory bowel disease, there were several examples where the putatively causal enhancer was only active in a particular immune cell or a stimulated state of an immune cell.

# My thoughts

What I like about this model is that it is so different to many of the integrative and machine learning methods we see in genomics. It uses two kinds of data, and relatively simple computations. There is no machine learning. There is no sequence evolution or conservation analysis. Instead, measure two relevant quantities, standardise and preprocess them a bit, and then multiply them together.

If the success of the activity-by-contact model for prioritising causal enhancers generalises beyond the 18 causative genes investigated in the original paper, this is an argument for simple biology-based heuristics over machine learning models. It also suggest that, in the absence of contact data, one might do well by prioritising variant in enhancers that are highly active in relevant cell types, and picking the closest gene as the proposed causative gene.

However, the dataset needs to cover the relevant cell types, and possibly cells that are in the relevant stimulated states, meaning that it provides a motivation for rich conditional atlas-style datasets of chromatin and chromosome conformation.

I am, personally, a little bit sad that expression QTL methods seem to doing poorly. On the other hand, it makes some sense that eQTL might not have the genomic resolution to connect enhancers to genes.

Finally, if the relatively simple activity-by-contact model or the ridiculously simple method of ”picking the closest gene” beats machine learning models using the same data types and more, it suggests that the machine learning methods might not be solving theright problem. After all, they are not trained directly to prioritise variants for complex traits — because there are too few known causative variants for complex traits.

Literature

Fulco, C. P., Nasser, J., Jones, T. R., Munson, G., Bergman, D. T., Subramanian, V., … & Engreitz, J. M. (2019). Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nature genetics.

Nasser, J., Bergman, D. T., Fulco, C. P., Guckelberger, P., Doughty, B. R., Patwardhan, T. A., … & Engreitz, J. M. (2021). Genome-wide enhancer maps link risk variants to disease genes. Nature.