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Lior Pachter

Division of Biology and Biological Engineering &

Department of Computing and Mathematical Sciences California Institute of Technology

**Abstract**

A recently published pilot study on the efficacy of 25-hydroxyvitamin D3 (calcifediol) in reducing ICU admission of hospitalized COVID-19 patients, concluded that the treatment “seems able to reduce the severity of disease, but larger trials with groups properly matched will be required go show a definitive answer”. In a follow-up paper, Jungreis and Kellis re-examine this so-called “Córdoba study” and argue that the authors of the study have undersold their results. Based on a reanalysis of the data in a manner they describe as “rigorous” and using “well established statistical techniques”, they urge the medical community to “consider testing the vitamin D levels of all hospitalized COVID-19 patients, and taking remedial action for those who are deficient.” Their recommendation is based on two claims: in an examination of unevenness in the distribution of one of the comorbidities between cases and controls, they conclude that there is “no evidence of incorrect randomization”, and they present a “mathematical theorem” to make the case that the effect size in the Córdoba study is significant to the extent that “they can be confident that if assignment to the treatment group had no effect, we would not have observed these results simply due to chance.”

Unfortunately, the “mathematical analysis” of Jungreis and Kellis is deeply flawed, and their “theorem” is vacuous. Their analysis cannot be used to conclude that the Córdoba study shows that calcifediol significantly reduces ICU admission of hospitalized COVID- 19 patients. Moreover, the Córdoba study is fundamentally flawed, and therefore there is nothing to learn from it.

**The Córdoba study**

The Córdoba study, described by the authors as a pilot, was ostensibly a randomized controlled trial, designed to determine the efficacy of 25-hydroxyvitamin D3 in reducing ICU admission of hospitalized COVID-19 patients. The study consisted of 76 patients hospitalized for COVID-19 symptoms, with 50 of the patients treated with calcifediol, and 26 not receiving treatment. Patients were administered “standard care”, which according to the authors consisted of “a combination of hydroxychloroquine, azithromycin, and for patients with pneumonia and NEWS score 5, a broad spectrum antibiotic”. Crucially, admission to the ICU was determined by a “Selection Committee” consisting of intensivists, pulmonologists, internists, and members of an ethics committee. The Selection Committee based ICU admission decisions on the evaluation of several criteria, including presence of comorbidities, and the level of dependence of patients according to their needs and clinical criteria.

The result of the Córdoba trial was that only 1/50 of the treated patients was admitted to the ICU, whereas 13/26 of the untreated patients were admitted (p-value = 7.7 ∗ 10^{−7} by Fisher’s exact test). This is a minuscule p-value but it is meaningless. Since there is no record of the Selection Committee deliberations, it impossible to know whether the ICU admission of the 13 untreated patients was due to their previous high blood pressure comorbidity. Perhaps the 11 treated patients with the comorbidity were not admitted to the ICU because they were older, and the Selection Committee considered their previous higher blood pressure to be more “normal” (14/50 treatment patients were over the age of 60, versus only 5/26 of the untreated patients).

Figure 1: Table 2 from [1] showing the comorbidities of patients. It is reproduced by virtue of [1] being published open access under the CC-BY license.

The fact that admission to the ICU could be decided in part based on the presence of co-morbidities, and that there was a significant imbalance in one of the comorbidities, immediately renders the study results meaningless. There are several other problems with it that potentially confound the results: the study did not examine the Vitamin D levels of the treated patients, nor was the untreated group administered a placebo. Most importantly, the study numbers were tiny, with only 76 patients examined. Small studies are notoriously problematic, and are known to produce large effect sizes [9]. Furthermore, sloppiness in the study does not lead to confidence in the results. The authors state that the “rigorous protocol” for determining patient admission to the ICU is available as Supplementary Material, but there is no Supplementary Material distributed with the paper. There is also an embarrassing typo: Fisher’s exact test is referred to twice as “Fischer’s test”. To err once in describing this classical statistical test may be regarded as misfortune; to do it twice looks like carelessness.

**A pointless statistics exercise**

The Córdoba study has not received much attention, which is not surprising considering that by the authors’ own admission it was a pilot that at best only motivates a properly matched and powered randomized controlled trial. Indeed, the authors mention that such a trial (the COVIDIOL trial), with data being collected from 15 hospitals in Spain, is underway. Nevertheless, Jungreis and Kellis [3], apparently mesmerized by the 7.7 ∗ 10^{−7} p-value for ICU admission upon treatment, felt the need to “rescue” the study with what amounts to faux statistical gravitas. They argue for immediate consideration of testing Vitamin D levels of hospitalized patients, so that “deficient” patients can be administered some form of Vitamin D “to the extent it can be done safely”. Their message has been noticed; only a few days after [3] appeared the authors’ tweet to promote it has been retweeted more than 50 times [8].

Jungreis and Kellis claim that the p-value for the effect of calcifediol on patients is so significant, that in and of itself it merits belief that administration of calcifediol does, in fact, prevent admission of patients to ICUs. To make their case, Jungreis and Kellis begin by acknowledging that imbalance between the treated and untreated groups in the previous high blood pressure comorbidity may be a problem, but claim that there is “no evidence of incorrect randomization.” Their argument is as follows: they note that while the p-value for the imbalance in the previous high blood pressure comorbidity is 0.0023, it should be adjusted for the fact that there are 15 distinct comorbidities, and that just by chance, when computing so many p-values, one might be small. First, an examination of Table 2 in [1] (Figure 1) shows that there were only 14 comorbidities assessed, as none of the patients had previous chronic kidney disease. Thus, the number 15 is incorrect. Second, Jungreis and Kellis argue that a Bonferroni correction should be applied, and that this correction should be based on 30 tests (=15 × 2). The reason for the factor of 2 is that they claim that when testing for imbalance, one should test for imbalance in both directions. By applying the Bonferroni correction to the p-values, they derive a “corrected” p-value for previous high blood pressure being imbalanced between groups of 0.069. They are wrong on several counts in deriving this number. To illustrate the problems we work through the calculation step-by-step:

The question we want to answer is as follows: given that there are multiple comorbidities, is there is a significant imbalance in *at least* one comorbidity. There are several ways to test for this, with the simplest being Šidák’s correction [10] given by

where *m* is the minimum p-value among the comorbidities, and *n* is the number of tests. Plugging in *m = 0.0023* (the smallest p-value in Table 2 of [1]) and *n = 14* (the number of comorbidities) one gets 0.032 (note that the Bonferroni correction used by Jungreis And Kellis is the Taylor approximation to the Šidák correction when *m* is small). The Šidák correction is based on an assumption that the tests are independent. However, that is certainly not the case in the Córdoba study. For example, having at least one prognostic factor is one of the comorbidities tabulated. In other words, the p-value obtained is conservative. The calculation above uses n = 14, but Jungreis and Kellis reason that the number of tests is 30 = 15 × 2, to take into account an imbalance in either the treated or untreated direction. Here they are assuming two things: that two-sided tests for each comorbidity will produce double the p-value of a one-sided test, and that two sided tests are the “correct” tests to perform. They are wrong on both counts. First, the two-sided Fisher exact test does not, in general produce a p-value that is double the 1-sided test. The study result is a good example: 1/49 treated patients admitted to the ICU vs. 13/26 untreated patients produces a p-value of 7.7 ∗ 10^{−7} for both the 1-sided and 2-sided tests. Jungreis and Kellis do not seem to know this can happen, nor understand why; they go to great lengths to explain the importance of conducting a 1-sided test for the study result. Second, there is a strong case to be made that a 1-sided test is the correct test to perform for the comorbidities. The concern is not whether there was an imbalance of any sort, but whether the imbalance would skew results by virtue of the study including too many untreated individuals with comorbidities. In any case, if one were to give Jungreis and Kellis the benefit of the doubt, and perform a two sided test, the corrected p-value for the previous high blood pressure comorbidity is 0.06 and not 0.069.

The most serious mistake that Jungreis and Kellis make, however, is in claiming that one can accept the null hypothesis of a hypothesis test when the p-value is greater than 0.05. The p-value they obtain is 0.069 which, even if it is taken at face value, is not grounds for claiming, as Jungreis and Kellis do, that “this is not significant evidence that the assignment was not random” and reason to conclude that there is “no evidence of incorrect randomization”. That is not how p-values work. A p-value less than 0.05 allows one to reject the null hypothesis (assuming 0.05 is the threshold chosen), but a p-value above the chosen threshold is not grounds for accepting the null. Moreover, the corrected p-value is 0.032 which is certainly grounds for rejecting the null hypothesis that the randomization was random.

Correction of the incorrect Jungreis and Kellis statistics may be a productive exercise in introductory undergraduate statistics for some, but it is pointless insofar as assessing the Córdoba study. While the extreme imbalance in the previous high blood pressure comorbidity is problematic because patients with the comorbidity may be more likely to get sick and require ICU admission, the study was so flawed that the exact p-value for the imbalance is a moot point. Given that the presence of comorbidities, not just their effect on patients, was a factor in determining which patients were admitted to the ICU, the extreme imbalance in the previous high blood pressure comorbidity renders the result of the study meaningless *ex facie*.

**A definition is not a theorem is not proof of efficacy**

In an effort to fend off criticism that the comorbidities of patients were improperly balanced in the study, Jungreis and Kellis go further and present a “theorem” they claim shows that there was a minuscule chance that an uneven distribution of comorbidities could render the study results not significant. The “theorem” is stated twice in their paper, and I’ve copied both theorem statements verbatim from their paper:

**Theorem 1** *In a randomized study, let p be the p-value of the study results, and let q be the probability that the randomization assigns patients to the control group in such a way that the values of P _{prognostic}(Patient) are sufficiently unevenly distributed between the treatment and control groups that the result of the study would no longer be statistically significant at the 95% level after p controlling for the prognostic risk factors. Then .*

According to Jungreis and Kellis, *P _{prognostic}(Patient)* is the following: “There can be any number of prognostic risk factors, but if we knew what all of them were, and their effect sizes, and the interactions among them, we could combine their effects into a single number for each patient, which is the probability, based on all known and yet-to-be discovered risk factors at the time of hospital admission, that the patient will require ICU care if not given the calcifediol treatment. Call this (unknown) probability

*P*.”

_{prognostic}(Patient)The theorem is restated in the Methods section of Jungreis and Kellis paper as follows:

**Theorem 2** *In a randomized controlled study, let p be the p-value of the study outcome, and let q be the probability that the randomization distributes all prognostic risk factors combined sufficiently unevenly between the treatment and control groups that when controlling for these prognostic risk p factors the outcome would no longer be statistically significant at the 95% level. Then .*

While it is difficult to decipher the language the “theorem” is written in, let alone its meaning (note Theorem 1 and Theorem 2 are supposedly the same theorem), I was able to glean something about its content from reading the “proof”. The mathematical content of whatever the theorem is supposed to mean, is the definition of conditional probability, namely that if A and B are events with , then

.

To be fair to Jungreis and Kellis, the “theorem” includes the observation that

This is not, by any stretch of the imagination, a “theorem”; it is literally the definition of conditional probability followed by an elementary inequality. The most generous interpretation of what Jungreis and Kellis were trying to do with this “theorem”, is that they were showing that the p-value for the study is so small, that it is small even after being multiplied by 20. There are less generous interpretations.

**Does Vitamin D intake reduce ICU admission?**

There has been a lot of interest in Vitamin D and its effects on human health over the past decade [2], and much speculation about its relevance for COVID-19 susceptibility and disease severity. One interesting result on disease susceptibility was published recently: in a study of 489 patients, it was found that the relative risk of testing positive for COVID-19 was 1.77 times greater for patients with likely deficient vitamin D status compared with patients with likely sufficient vitamin D status [7]. However, definitive results on Vitamin D and its relationship to COVID- 19 will have to await larger trials. One such trial, a large randomized clinical trial with 2,700 individuals sponsored by Brigham and Women’s Hospital, is currently underway [4]. While this study might shed some light on Vitamin D and COVID-19, it is prudent to keep in mind that the outcome is not certain. Vitamin D levels are confounded with many socioeconomic factors, making the identification of causal links difficult. In the meantime, it has been suggested that it makes sense for individuals to maintain reference nutrient intakes of Vitamin D [6]. Such a public health recommendation is not controversial.

As for Vitamin D administration to hospitalized COVID-19 patients reducing ICU admission, the best one can say about the Córdoba study is that nothing can be learned from it. Unfortunately, the poor study design, small sample size, availability of only summary statistics for the comorbidities, and imbalanced comorbidities among treated and untreated patients render the data useless. While it may be true that calcifediol administration to hospital patients reduces subsequent ICU admission, it may also not be true. Thus, the follow-up by Jungreis and Kellis is pointless at best. At worst, it is irresponsible propaganda, advocating for potentially dangerous treatment on the basis of shoddy arguments masked as “rigorous and well established statistical techniques”. It is surprising to see Jungreis and Kellis argue that it may be unethical to conduct a placebo randomized controlled trial, which is one of the most powerful tools in the development of safe and effective medical treatments. They write “the ethics of giving a placebo rather than treatment to a vitamin D deficient patient with this potentially fatal disease would need to be evaluated.” The evidence for such a policy is currently non-existent. On the other hand, there are plenty of known risks associated with excess Vitamin D [5].

**References**

- Marta Entrenas Castillo, Luis Manuel Entrenas Costa, José Manuel Vaquero Barrios, Juan Francisco Alcalá Díaz, José López Miranda, Roger Bouillon, and José Manuel Quesada Gomez. Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study.
*The Journal of steroid biochemistry and molecular biology,*203:105751, 2020. - Michael F Holick. Vitamin D deficiency.
*New England Journal of Medicine*, 357(3):266–281, 2007. - Irwin Jungreis and Manolis Kellis. Mathematical analysis of Córdoba calcifediol trial suggests strong role for Vitamin D in reducing ICU admissions of hospitalized COVID-19 patients.
*medRxiv*, 2020. - JoAnn E Manson. https://clinicaltrials.gov/ct2/show/nct04536298.
- Ewa Marcinowska-Suchowierska, Małgorzata Kupisz-Urbańska, Jacek Łukaszkiewicz, Paweł Płudowski, and Glenville Jones. Vitamin D toxicity–a clinical perspective.
*Frontiers in endocrinology*, 9:550, 2018 - Adrian R Martineau and Nita G Forouhi. Vitamin D for COVID-19: a case to answer?
*The Lancet Diabetes & Endocrinology*, 8(9):735–736, 2020. - David O Meltzer, Thomas J Best, Hui Zhang, Tamara Vokes, Vineet Arora, and Julian Solway. Association of vitamin D status and other clinical characteristics with COVID-19 test results.
*JAMA network open*, 3(9):e2019722–e2019722, 2020. - Vivien Shotwell.
*https://tweetstamp.org/1327281999137091586*. - Robert Slavin and Dewi Smith. The relationship between sample sizes and effect sizes in systematic reviews in education.
*Educational evaluation and policy analysis*, 31(4):500–506, 2009. - Lynn Yi, Harold Pimentel, Nicolas L Bray, and Lior Pachter. Gene-level differential analysis at transcript-level resolution.
*Genome biology*, 19(1):53, 2018.

Five years ago on this day, Nicolas Bray and I wrote a blog post on The network nonsense of Manolis Kellis in which we described the paper Feizi et al. 2013 from the Kellis lab as dishonest and fraudulent. Specifically, we explained that:

**“Feizi et al. have written a paper that appears to be about inference of edges in networks based on a theoretically justifiable model but**

**the method used to obtain the results in the paper is completely different than the idealized version sold in the main text of the paper and****the method actually used has parameters that need to be set, yet no approach to setting them is provided. Even worse,****the authors appear to have deliberately tried to hide the existence of the parameters. It looks like****the reason for covering up the existence of parameters is that the parameters were tuned to obtain the results.****Moreover,****the results are not reproducible. The provided data and software is not enough to replicate even a single figure in the paper. This is disturbing because****the performance of the method on the simplest of all examples, a correlation matrix arising from a Gaussian graphical model, is poor.”**

A second point we made is that the justification for the method, which the authors called “network deconvolution” was nonsense. For example, the authors wrote that **“The model assumes that networks are “l****inear time-invariant flow-preserving operators.”** Perhaps I take things too literally when I read papers but I have to admit that five years later I still don’t understand the sentence. However just because a method is ad-hoc, heuristic, or perhaps poorly explained, doesn’t mean it won’t work well in practice. In the blog post we compared network deconvolution to regularized partial correlation on simulated data, and found network deconvolution performed poorly. But in a responding comment, Kellis noted that “in our experience, partial correlation performed very poorly in practice.” He added that “We have heard very positive feedback from many other scientists using our software successfully in diverse applications.”

Fortunately we can now evaluate Kellis’ claims in light of an independent analysis in Wang, Pourshafeie, Zitnik et al. 2018, a paper from the groups of Serafim Batzoglou and Jure Leskovec (in collaboration with Carlos Bustamante) at Stanford University. There are three main results presented in Wang, Pourshafeie and Zitnik et al. 2018 that summarize the benchmarking of network deconvolution and other methods, and I reproduce figures showing the results below. The first shows the performance of network deconvolution and some other network denoising methods on a problem of butterfly species identification (network deconvolution is abbreviated ND and is shown in green). RAW (in blue) is the original unprocessed network. **Network deconvolution is much worse than RAW**:

The second illustrates the performance of network denoising methods on Hi-C data. The performance metric in this case is normalized mutual information (NMI) which Wang, Pourshafeie, Zitnik et al. described as “a fair representation of overall performance”. **Network deconvolution (ND, dark green) is again worse than RAW (dark blue)**:

Finally, in an analysis of gene function from tissue-specific gene interaction networks, ND (blue) does perform better than RAW (pink) although barely. In four cases out of eight shown it is the worst of the four methods benchmarked:

Network deconvolution was claimed to be applicable to any network when it was published. At the time, Feizi stated that “We applied it to gene networks, protein folding, and co-authorship social networks, but our method is general and applicable to many other network science problems.” A promising claim, but in reality it is difficult to beat the nonsense law: **Nonsense methods tend to produce nonsense results**.

The Feizi et al. 2013 paper now has 178 citations, most of them drive by citations. Interestingly this number, 178 is **exactly** the number of citations of the Barzel et al. 2013 network nonsense paper, which was published in the same issue of Nature Biotechnology. Presumably this reflects the fact that authors citing one paper feel obliged to cite the other. These pair of papers were thus an impact factor win for the journal. For the first authors on the papers, the network deconvolution/silencing work is their most highly cited first author papers respectively. Barzel is an assistant professor at Bar-Ilan University where he links to an article about his network nonsense on his “media page”. Feizi is an assistant professor at the University of Maryland where he lists Feizi et al. 2013 among his “selected publications“. Kellis teaches the “network deconvolution” and its associated nonsense in his computational biology course at MIT. And why not? These days truth seems to matter less and less in every domain. A statement doesn’t have to be true, it just has to work well on YouTube, Twitter, Facebook, or some webpage, and as long as some people believe it long enough, say until the next grant cycle, promotion evaluation, or election, then what harm is done? A win-win for everyone. Except science.

Two weeks ago in my post Pachter’s P-value Prize I offered ** for justifying a reasonable null model and a p-value (***p)* associated to the statement “”**Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair, providing strong support for a specific model of evolution, and allowing us to distinguish ancestral and derived functions**” in the paper

M. Kellis, B.W. Birren and E.S. Lander, Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisae, *Nature* 2004 (hereafter referred to as the KBL paper).

Today I am happy to announce the winner of the prize. But first, I want to thank the many readers of the blog who offered comments (>135 in total) that are extraordinary in their breadth and depth, and that offer a glimpse of what scientific discourse can look like when not restricted to traditional publishing channels. You have provided a wonderful public example of what “peer review” should mean. Coincidentally, and to answer one of the questions posted, the blog surpassed one million views this past Saturday (the first post was on August 19th, 2013), a testament to the the fact that the collective peer reviewing taking place on these pages is not only of very high quality, but also having an impact.

I particularly want to thank the students who had the courage to engage in the conversation, and also faculty who published comments using their name. In that regard, I admire and commend Joshua Plotkin and Hunter Fraser for deciding to deanonymize themselves by agreeing to let me announce here that they were the authors of the critique sent to the authors in April 2004 initially posted as an anonymous comment on the blog.

The discussion on the blog was extensive, touching on many interesting issues and I only summarize a few of the threads of discussion here. I decided to touch on a number of key points made in order to provide context and justification for my post and selection of the prize winner.

**The value of post-publication review**

One of the comments made in response to my post that I’d like to respond to first was by an author of KBL who dismissed the entire premise of the my challenge writing “We can keep debating this after 11 years, but I’m sure we all have much more pressing things to do (grants? papers? family time? attacking 11-year-old papers by former classmates? guitar practice?)”

This comment exemplifies the proclivity of some authors to view publication as the encasement of work in a casket, buried deeply so as to never be opened again lest the skeletons inside it escape. But is it really beneficial to science that much of the published literature has become, as Ferguson and Heene noted, a vast graveyard of undead theories? Surely the varied and interesting comments posted in response to my challenge (totaling >25,000 words and 50 pages in Arial 11 font), demonstrate the value of communal discussion of science *after* publication.

For the record, this past month I did submit a paper and also a grant, and I did spend lots of time with my family. I didn’t practice the guitar but I did play the piano. Yet in terms of research, for me the highlight of the month was reading and understanding the issues raised in the comments to my blog post. Did I have many other things to do? Sure. But what is more pressing than *understanding* if the research one does is to be meaningful?

**The null model**

A few years ago I introduced a new two-semester freshman math course at UC Berkeley for intended biology majors called Math 10- Methods of Mathematics: Calculus, Statistics and Combinatorics“. One of the key ideas we focus on in the first semester is that of a *p*-value. The idea of measuring significance of a biological result via a statistical computation involving probabilities is somewhat unnatural, and feedback from the students confirms what one might expect: that the topic of *p*-values is among the hardest in the course. Math for biologists turns out to be much harder than calculus. I believe that at Berkeley we are progressive in emphasizing the importance of statistics for biology majors at the outset of their education (to be clear, this is a recent development). The prevailing state is that of statistical illiteracy, and the result is that *p*-values are frequently misunderstood, abused, and violated in just about every possible way imaginable (see, e.g., here, here and here).

P-values require a null hypothesis and a test statistic, and of course one of the most common misconceptions about them is that when they are large they confirm that the null hypothesis is correct. No! And worse, a small *p*-value cannot be used to accept an alternative to the null, only to (confidently) reject the null itself. And rejection of the null comes with numerous subtle issues and caveats (see arguments against the *p-*value in the papers mentioned above). So what is the point?

I think the KBL paper makes for an interesting case study of when *p-*values can be useful. For starters, the construction of a null model is already a useful exercise, because it is a thought experiment designed to test ones understanding of the problem at hand. The senior author of the KBL paper argues that “we were interested in seeing whether, for genes where duplication frees up at least one copy to evolve rapidly, the evidence better fits one model (“Ohno”: only one copy will evolve quickly) or an alternative model (both genes will evolve quickly).” While I accept this statement at face value, it is important to acknowledge that if there is any science to data science, it is the idea that when examining data one must think beyond the specific hypotheses being tested and consider alternative explanations. This is the essence of what my colleague Ian Holmes is saying in his comment. In data analysis, thinking outside of the box (by using statistics) is not optional. If one is lazy and resorts to intuition then, as Páll Melted points out, one is liable to end up with fantasy.

The first author of KBL suggests that the “paper was quite explicit about the null model being tested.” But I was unsure of whether to assume that the one-gene-only-speeds-up model is the null based on”we sought to distinguish between the Ohno one-gene-only speeds-up (OS) model and the alternative both-genes speed-up (BS) model” or was the null the BS model because “the Ohno model is 10^87 times more likely, leading to significant rejection of the BS null”? Or was the paper being explicit about not having a null model at all, because “Two alternatives have been proposed for post-duplication”, or was it the opposite, i.e. two null models: “the OS and BS models are each claiming to be right 95% of the time”? I hope I can be forgiven for failing, despite trying very hard, to identify a null model in either the KBL paper, or the comments of the authors to my blog.

There is however a reasonable null model, and it is the “independence model”, which to be clear, is the model where each gene after duplication “accelerates” independently with some small probability (80/914). The suggestions that “the independence model is not biologically rooted” or that it “would predict that only 75% of genes would be preserved in at least one copy, and that 26% would be preserved in both copies” are of course absurd, as explained by Erik van Nimwegen who explains why point clearly and carefully. The fact that many entries reached the same conclusion about the suitable null model in this case is reassuring. I think it qualifies as a “reasonable model” (thereby passing the threshold for my prize).

**The p-value**

One of my favorite missives about p-values is by Andrew Gelman, who in “P-values and statistical practice” discusses the subtleties inherent in the use and abuse of p-values. But as my blog post illustrates, subtlety is one thing, and ignorance is an entirely different matter. Consider for example, the entry by Manolis Kellis who submitted that thus claiming that I owe him 903,659,165 million billion trillion quadrillion quintillion sextillion dollars (even more than the debt of the United States of America). His entry will not win the prize, although the elementary statistics lesson that follows is arguably worth a few dollars (for him). First, while it is true that a p-value can be computed from the (log) likelihood ratio when the null hypothesis is a special case of the alternative hypothesis (using the chi^2 distribution), the ratio of two likelihoods is not a *p*-value! Probabilities of events are also not *p*-values! For example, the comment that “I calculated p-values for the exact count, but the integral/sum would have been slightly better” is a non-starter. Even though KBL was published in 2004, this is apparently the level of understanding of *p*-values of one of the authors, a senior computational biologist and professor of computer science at MIT in 2015. Wow.

So what is “the correct” *p-*value? It depends, of course, on the test statistic. Here is where I will confess that like many professors, I had an answer in mind before asking the question. I was thinking specifically of the setting that leads to 0.74 (or 0.72/0.73, depending on roundoff and approximation). Many entries came up with the same answer I had in mind and therefore when I saw them I was relieved: I owed $135, which is what I had budgeted for the exercise. **I was wrong**. The problem with the answer 0.74 is that it is the answer to the specific question: what is the probability of seeing 4 or less pairs accelerate out of 76 pairs in which at least one accelerated. A better test statistic was proposed by Pseudo in which he/she asked for the probability of seeing 5% or less of the pairs accelerate from among the pairs with at least one gene accelerating when examining data from the null model with 457 pairs. This is a subtle but important distinction, and provides a stronger result (albeit with a smaller *p*-value). The KBL result is not striking even forgoing the specific numbers of genes measured to have accelerated in at least one pair (of course just because *p=0.64* also does not mean the independence model is correct). What it means is that the data as presented simply weren’t “striking”.

One caveat in the above analysis is that the arbitrary threshold used to declare “acceleration” is problematic. For example, one might imagine that other thresholds produce more convincing results, i.e. farther from the null, but of course even if that were true the use of an arbitrary cutoff was a poor approach to analysis of the data. Fortunately, regarding the specific question of its impact in terms of the analysis, we do not have to imagine. Thanks to the diligent work of Erik van Nimwegen, who went to the effort of downloading the data and reanalyzing it with different thresholds (from 0.4 to 1.6), we know that the null cannot be rejected even with other thresholds.

**The award**

There were many entries submitted and I read them all. My favorite was by Michael Eisen for his creative use of multiple testing correction, although I’m happier with the direction that yields $8.79. I will not be awarding him the prize though, because his submission fails the test of “reasonable”, although I will probably take him out to lunch sometime at Perdition Smokehouse.

I can’t review every single entry or this post, which is already too long, would become unbearable, but I did think long and hard about the entry of K. It doesn’t directly answer the question of why the 95% number is striking, nor do I completely agree with some of the assumptions (e.g. if neither gene in a pair accelerates then the parent gene was not accelerated pre-WGD). But I’ll give the entry an honorable mention.

The prize will be awarded to Pseudo for defining a reasonable null model and test statistic, and producing the smallest *p*-value within that framework. With a *p*-value of *0.64 *I will be writing a check in the amount of $156.25. Congratulations Pseudo!!

**The biology**

One of the most interesting results of the blog post was, in my opinion, the extensive discussion about the truth. Leaving aside the flawed analysis of KBL, what is a reasonable model for evolution post-WGD? I am happy to see the finer technical details continue to be debated, and the intensity of the conversation is awesome! Pavel Pevzner’s cynical belief that “science fiction” is not a literary genre but rather a description of what is published in the journal Science may be realistic, but I hope the comments on my blog can change his mind about what the future can look like.

In lieu of trying to summarize the scientific conversation (frankly, I don’t think I could do justice to some of the intricate and excellent arguments posted by some of the population geneticists) I’ll just leave readers to enjoy the comment threads on their own. Comments are still being posted, and I expect the blog post to be a “living” post-publication review for some time. May the skeletons keep finding a way out!

**The importance of wrong**

Earlier in this post I admitted to being wrong. I have been wrong many times. Even though I’ve admitted some of my mistakes on this blog and elsewhere in talks, I would like to joke that I’m not going to make it easy for you to find other flaws in my work. That would be a terrible mistake. Saying “I was wrong” is important for science and essential for scientists. Without it people lose trust in both.

I have been particularly concerned with a lack of “I was wrong” in genomics. Unfortunately, there is a culture that has developed among “leaders” in the field where the three words admitting error or wrongdoing are taboo. The recent paper of Lin *et al.* critiqued by Gilad-Mizrahi is a good example. Leaving aside the question of whether the result in the paper is correct (there are strong indications that it isn’t), Mizrahi-Gilad began their critique on twitter by noting that the authors had completely failed to account for, or even discuss, batch effect. Nobody, and I mean nobody who works on RNA-Seq would imagine for even a femtosecond that this is ok. It was a major oversight and mistake. The authors, any of them really, could have just come out and said “I was wrong”. Instead, the last author on the paper, Mike Snyder, told reporters that “All of the sequencing runs were conducted by the same person using the same reagents, lowering the risk of unintentional bias”. Seriously?

Examples abound. The “ENCODE 80% kerfuffle” involved claims that “80% of the genome is functional”. Any self-respecting geneticist recognizes such headline grabbing as rubbish. Ewan Birney, a distinguished scientist who has had a major impact on genomics having being instrumental in the ENSEMBL project and many other high-profile bioinformatics programs defended the claim on BBC:

“EB: Ah, so, I don’t — It’s interesting to reflect back on this. For me, the big important thing of ENCODE is that we found that a lot of the genome had some kind of biochemical activity. And we do describe that as “biochemical function”, but that word “function” in the phrase “biochemical function”is the thing which gets confusing. If we use the phrase “biochemical activity”, that’s precisely what we did, we find that the different parts of the genome, [??] 80% have some specific biochemical event we can attach to it. I was often asked whether that 80% goes to 100%, and that’s what I believe it will do. So, in other words, that number is much more about the coverage of what we’ve assayed over the entire genome. In the paper, we say quite clearly that the majority of the genome is not under negative selection, and we say that most of the elements are not under pan-mammalian selection. So that’s negative selection we can detect between lots of different mammals. [??] really interesting question about what is precisely going on in the human population, but that’s — you know, I’m much closer to the instincts of this kind of 10% to 20% sort of range about what is under, sort of what evolution cares about under selection.”

This response, and others by members of the ENCODE consortium upset many people who may struggle to tell apart white and gold from blue and black, but certainly know that white is not black and black is not white. Likewise, I suspect the response of KBL to my post disappointed many as well. For Fisher’s sake, why not just acknowledge what is obvious and true?

**The personal critique of professional conduct**

A conversation topic that emerged as a result of the blog (mostly on other forums) is the role of style in online discussion of science. Specifically, the question of whether personal attacks are legitimate has come up previously on my blog pages and also in conversations I’ve had with people. Here is my opinion on the matter:

Science is practiced by human beings. Just like with any other human activity, some of the humans who practice it are ethical while others are not. Some are kind and generous while others are… not. Occasionally scientists are criminal. Frequently they are honorable. Of particular importance is the fact that most scientists’ behavior is not at any of these extremes, but rather a convex combination of the mentioned attributes and many others.

In science it is people who benefit, or are hurt, by the behavior of scientists. Preprints on the bioRxiv do not collect salaries, the people who write them do. Papers published in journals do not get awarded or rejected tenure, people do. Grants do not get jobs, people do. The behavior of people in science affects… *people*.

Some argue for a de facto ban on discussing the personal behavior of scientists. I agree that the personal *life* of scientists is off limits. But their professional life shouldn’t be. When Bernie Madoff fabricated gains of $65 billion it was certainly legitimate to criticize him *personally. *Imagine if that was taboo, and instead only the technical aspects of his Ponzi scheme were acceptable material for public debate. That would be a terrible idea for the finance industry, and so it should be for science. Science is not special among the professions, and frankly, the people who practice it hold no primacy over others.

I therefore believe it is not only acceptable but imperative to critique the *professional* behavior of persons who are scientists. I also think that doing so will help eliminate the problematic devil–saint dichotomy that persists with the current system. Having developed a culture in which personal criticism is outlawed in scientific conversations while only science is fair fodder for public discourse, we now have a situation where scientists are all presumed to be living Gods, or else serious criminals to be outlawed and banished from the scientific community. Acknowledging that there ought to be a grey zone, and developing a healthy culture where critique of all aspects of science and scientists is possible and encouraged would relieve a lot of pressure within the current system. It would also be more fair and just.

**A final wish**

I wish the authors of the KBL paper would publish the reviews of their paper on this blog.

In this blog post **I offer a cash prize** for computing a *p*-value [update June 9th: the winner has been announced!]. For details about the competition you can skip directly to the challenge. But context is important:

**Background**

I’ve recently been reading a bioRxiv posting by X. Lan and J. Pritchard, Long-term survival of duplicate genes despite absence of subfunctionalized expression (2015) that examines the question of whether gene expression data (from human and mouse tissues) supports a model of duplicate preservation by subfunctionalization.

The term *subfunctionalization*** **is a hypothesis for explaining the ubiquity of persistence of gene duplicates in extant genomes. The idea is that gene pairs arising from a duplication event evolve, via neutral mutation, different functions that are distinct from their common ancestral gene, yet together recapitulate the original function. It was introduced in 1999 an alternative to the older hypothesis of *neofunctionalization*, which posits that novel gene functions arise by virtue of “retention” of one copy of a gene after duplication, while the other copy morphs into a new gene with a new function. Neofunctionalization was first floated as an idea to explain gene duplicates in the context of evolutionary theory by Haldane and Fisher in the 1930s, and was popularized by Ohno in his book Evolution by Gene Duplication published in 1970. The cartoon below helps to understand the difference between the *functionalization hypotheses (adapted from wikipedia):

Lan and Pritchard examine the credibility of the sub- and neofunctionalization hypotheses using modern high-throughput gene expression (RNA-Seq) data: in their own words “Based on theoretical models and previous literature, we expected that–aside from the youngest duplicates–most duplicate pairs would be functionally distinct, and that the primary mechanism for this would be through divergent expression profiles. In particular, the sub- and neofunctionalization models suggest that, for each duplicate gene, there should be at least one tissue where that gene is more highly expressed than its partner.”

What they found was that, in their words, that “surprisingly few duplicate pairs show any evidence of sub-/neofunctionalization of expression.” The went further, stating that “the prevailing model for the evolution of gene duplicates holds that, to survive, duplicates must achieve non-redundant functions, and that this usually occurs by partitioning the expression space. However, we report here that sub-/neofunctionalization of expression occurs extremely slowly, and generally does not happen until the duplicates are separated by genomic rearrangements. Thus, in most cases long-term survival must rely on other factors.” They propose instead that “following duplication the expression levels of a gene pair evolve so that their combined expression matches the optimal level. Subsequently, the relative expression levels of the two genes evolve as a random walk, but do so slowly (33) due to constraint on their combined expression. If expression happens to become asymmetric, this reduces functional constraint on the minor gene. Subsequent accumulation of missense mutations in the minor gene may provide weak selective pressure to eventually eliminate expression of this gene, or may free the minor gene to evolve new functions.”

The Lan and Pritchard paper is the latest in a series of works that examine high-browed evolutionary theories with hard data, and that are finding reality to be far more complicated than the intuitively appealing, yet clearly inadequate, hypotheses of neo- and subfunctionalization. One of the excellent papers in the area is

Dean *et al.* Pervasive and Persistent Redundancy among Duplicated Genes in Yeast, *PLoS Genetics*, 2008.

where the authors argue that in yeast “duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.” I mention this paper, from the Petrov lab, because its results are fundamentally at odds with what is arguably the first paper to provide genome-wide evidence *for *neofunctionalization (also in yeast):

M. Kellis, B.W. Birren and E.S. Lander, Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisae, *Nature* 2004.

At the time, the Kellis-Birren-Lander paper was hailed as containing “work that may lead to better understanding of genetic diseases” and in the press release Kellis stated that “understanding the dynamics of genome duplication has implications in understanding disease. In certain types of cancer, for instance, cells have twice as many chromosomes as they should, and there are many other diseases linked to gene dosage and misregulation.” He added that “these processes are not much different from what happened in yeast.” and the author of the press releases added that “whole genome duplication may have allowed other organisms besides yeast to achieve evolutionary innovations in one giant leap instead of baby steps. It may account for up to 80 percent (seen this number before?) of flowering plant species and could explain why fish are the most diverse of all vertebrates.”

This all brings me to:

In the abstract of their paper, Kellis, Birren and Lander wrote that:

“**Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair, providing strong support for a specific model of evolution, and allowing us to distinguish ancestral and derived functions.**” [boldface by authors]

In the main text of the paper, the authors expanded on this claim, writing:

“Strikingly, in nearly every case (95%), accelerated evolution was confined to only one of the two paralogues. This strongly supports the model in which one of the paralogues retained an ancestral function while the other, relieved of this selective constraint, was free to evolve more rapidly”.

The word “strikingly” suggests a result that is surprising in its statistical significance with respect to some null model the authors have in mind. The data is as follows:

The authors identified 457 duplicated gene pairs that arose by whole genome duplication (for a total of 914 genes) in yeast. Of the 457 pairs 76 showed accelerated (protein) evolution in *S. cerevisiae. *The term “accelerated” was defined to relate to amino acid substitution rates in *S. cerevisiae,* which were required to be 50% faster than those in another yeast species, *K. waltii*. Of the 76 genes, only four pairs were accelerated in *both* paralogs. Therefore 72 gene pairs showed acceleration in only one paralog (72/76 = 95%).

So, is it indeed “striking” that “in nearly every case (95%), accelerated evolution was confined to only one of the two praralogues”? Well, the authors don’t provide a *p**–*value in their paper, nor do they propose a null model with respect to which the question makes sense. So I am offering a prize to help crowdsource what should have been an exercise undertaken by the authors, or if not a requirement demanded by the referees. To incentivize people in the right direction,

**I will award **

to the person who can best justify a reasonable null model, together with a p-value (*p*) for the phrase “**Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair**” in the abstract of the Kellis-Birren-Lander paper. Notice the smaller the (justifiable) *p-*value someone can come up with, the larger the prize will be.

**Bonus**: explain in your own words how you think the paper was accepted to Nature without the authors having to justify their use of the word “strikingly” for a main result of the paper, and in a timeframe consisting of submission on December 17th 2003 (just three days before Hanukkah and one week before Christmas) and acceptance January 19th 2004 (Martin Luther King Jr. day).

**Rules**

To be eligible for the prize entries must be submitted as comments on this blog post by **11:59pm EST on Sunday May 31st June 7th, 2015** and they must be submitted with a valid e-mail address. I will keep the name (and e-mail address) of the winner anonymous if they wish (this can be ensured by using a pseudonym when submitting the entry as a comment). The prize, if awarded, will go to the person submitting the most complete, best explained solution that has a

*p*

*–*value calculation that is correct according to the model proposed. Preference will be given to submission from students, especially undergraduates, but individuals in any stage of their career, and from anywhere in the world, are encouraged to submit solutions. I reserve the right to interpret the phrase “reasonable null model” in a way that is consistent with its use in the scientific community and I reserve the right to not award the prize if no good/correct solutions are offered. Participants do not have to answer the bonus question to win.

Reproducibility has become a major issue in scientific publication, it is under scrutiny by many, making headlines in the news, is on the minds of journals, and there are guidelines for achieving it. Reproducibility is certainly important for scientific research, but I think that lost in the debate is the notion of *usability*. Reproducibility only ensures that the results of a paper can be recreated by others, but usability ensures that researchers can build on scientific work, and explore hypotheses and ideas unforeseen by authors of the original papers. Here I describe a case study in reproducibility and usability, that emerged from a previous post I wrote about the paper

- Soheil Feizi, Daniel Marbach, Muriel Médard & Manolis Kellis, Network deconvolution as a general method to distinguish direct dependencies in networks,
*Nature Biotechnology*31(8), 2013, p 726–733.

Feizi *et al. *describe a method called network deconvolution, that they claim improves the inference results for 8 out of 9 network inference methods, out of the 35 that were tested in the DREAM5 challenge. In DREAM5, participants were asked to examine four chip-based *gene x expression* matrices, and were also provided a list of transcription factors for each. They were then asked to provide ranked lists of transcription factor – gene interactions for each of the four datasets. The four datasets consisted of one computer simulation (the “*in silico*” dataset) and expression measurements in *E. coli, S. cerevisiae *and *S. aureus. *The consortium received submission from 29 different groups and ran 6 other “off-the-shelf” methods, while also developing its own “community” method (for a total of 36=29+6+1). The community method consisted of applying the Borda count to the 35 methods being tested, to produce a new consensus, or community, network. Nicolas Bray and I tried to replicate the results of Feizi *et al. *so that we could test for ourselves the performance of network deconvolution with different parameters and on other DREAM5 methods ( Feizi *et al.* tested only 9 methods; there were 36 in total). But despite contacting the authors for help we were unable to do so. In desperation, I even offered $100 for someone to replicate all of the figures in the paper. Perhaps as a result of my blogging efforts, or possibly due to a spontaneous change of heart, the authors finally released *some* of the code and data needed to reproduce *some* of the figures in their paper. In particular, I am pleased to say that the released material is sufficient to almost replicate Figure 2 of their paper which describes their results on a portion of the DREAM5 data. I say almost because the results for one of the networks is off, but to the authors credit it does appear that the distributed data and code are close to what was used to produce the figure in the paper (note: there is still not enough disclosure to replicate *all* of the figures of the paper, including the suspicious Figure S4 before and after revision of the supplement, and I am therefore not yet willing to concede the $100). What Feizi *et al.* did accomplish was to make their methods *usable*. That is to say, with the distributed code and data I was able to test the method with different parameters and on new datasets. In other words, **Feizi et al. is still not completely reproducible, but it is usable. **In this post, I’ll demonstrate why usability is important, and make the case that it is too frequently overlooked or confused with reproducibility. With usable network deconvolution code in hand, I was finally able to test some of the claims of Feizi

*et al.*First, I identify the relationship between the DREAM methods and the methods Feizi

*et al.*applied network deconvolution to. In the figure below, I have reproduced Figure 2 from Feizi

*et al.*together with Figure 2 from Marbach

*et al.:*

Figure 2 from Feizi *et al.* aligned to Figure 2 from Marbach *et al.*

The mapping is more complex than appears at first sight. For example, in the case of Spearman correlation (method Corr #2 in Marbach *et al., *method #5 in Feizi *et al.*), Feizi *et al.* ran network deconvolution on the method after taking absolute values. This makes no sense, as throwing away the sign is to throw away a significant amount of information, not to mention it destroys any hope of connecting the approach to the intuition of inferring directed interactions from the observed via the idealized “model” described in the paper. On the other hand, Marbach *et al.* evaluated Spearman correlation *with* sign. Without taking the absolute value before evaluation negative edges, strong (negative) interactions, are ignored. This is the reason for the very poor performance of Spearman correlation and the reason for the discrepancy in bar heights between Marbach *et al.** *and Feizi *et al.* for that method. The caption of Figure 2 in Feizi *et al.* begins “Network deconvolution applied to the inferred networks of top-scoring methods [1] from DREAM5..” This is obviously not true. Moreover, one network they did not test on was the community network of Marbach *et al.* which was the best method and the point of the whole paper. However the methods they did test on were ranked 2,3,4,6,8,12,14,16,28 (out of 36 methods). The 10th “community” method of Feizi *et al.* is actually the result of applying the community approach to the ND output from all the methods, so it is not in and of itself a test of ND. Of the nine tested methods, arguably only a handful were “top” methods. I do think its sensible to consider “top” to be the best methods for each category (although Correlation is so poor I would discard it altogether). That leaves four top methods. So instead of creating the illusion that network deconvolution improves 9/10 top scoring methods, what Feizi *et al.* should have reported is that **3 out of 4 of the top methods that were tested were improved by network deconvolution**. That is the result of running network deconvolution with the default parameters. I was curious what happens when using the parameters that Feizi *et al.* applied to the protein interaction data (alpha=1, beta=0.99). Fortunately, because they have made the code usable, I was able to test this. The overall result as well as the scores on the individual datasets are shown below: The Feizi *et al. *results on gene regulatory networks using parameters different from the default. The results are very different. Despite the claims of Feizi *et al.* that network deconvolution is robust to choice of parameters,** now only 1 out of 4 of the top methods are improved by network deconvolution**. Strikingly, the top three methods tested have their quality degraded. In fact, the top method in two out of the three datasets tested is made worse by network deconvolution. **Network deconvolution is certainly not robust to parameter choice. **What was surprising to me was the *improved* performance of network deconvolution on the *S. cerevisae* dataset, especially for the mutual information and correlation methods. In fact, the improvement of network deconvolution over the methods is appears extraordinary. At this point I started to wonder about what the improvements really mean, i.e. what is the “score” that is being measured. The y-axis, called the “score” by Feizi *et al.* and Marbach *et al.* seemed to be changing drastically between runs. I wondered… what exactly is the score? What do the improvements mean? It turns out that “score” is defined as follows:

.

This formula requires some untangling: First of all, AUROC is shorthand for area under the ROC (receiver operator curve), and AUPR for area under the PR (precision recall) curve. For context, ROC is a standard concept in engineering/statistics. Precision and recall are used frequently, but the PR curve is used much less than ROC . Both are measures for judging the quality of a binary classifier. In the DREAM5 setting, this means the following: there is a gold standard of “positives”, namely a set of edges in a network that should be predicted by a method, and the remainder of the edges will be considered “negatives”, i.e. they should not be predicted. A method generates a list of edges, sorted (ranked) in some way. As one proceeds through the list, one can measure the fraction of positives and false positives predicted. The ROC and PR curves measure the performance. A ROC is simply a plot showing the true positive rate for a method as a function of the false positive rate. Suppose that there are *m *positives in the gold standard out of a goal of *n *edges. If one examines the top *k* predictions of a method, then among them there will be *t** “*true” positives as well as *k**-t “*false” positives. This will result in a single point on the ROC, i.e. the point (). This can be confusing at first glance for a number of reasons. First, the points do not necessarily form a function, e.g. there can be points with the same *x-*coordinate. Second, as one varies *k *one obtains a set of points, not a curve. The ROC is a *curve,* and* *is obtained by taking the envelope of all of the points for . The following intuition is helpful in understanding ROC:

- The
*x*coordinate in the ROC is the false positive rate. If one doesn’t make any predictions of edges at all, then the false positive rate is 0 (in the notation above*k=0, t=0).*On the other hand, if all edges are considered to be “true”, then the false positive rate is 1 and the corresponding point on the ROC is (1,1), which corresponds to*k=n, t=m*. - If a method has no predictive power, i.e. the ranking of the edges tells you nothing about which edges really are true, then the ROC is the line
*y=x.*This is because lack of predictive power means that truncating the list at any*k*, results in the same proportion of true positives above and below the*k*th edge. And a simple calculation shows that this will correspond to the point () on the ROC curve. - ROC curves can be summarized by a single number that has meaning: the area under the ROC (AUROC). The observation above means that a method without any predictive power will have an AUROC of 1/2. Similarly, a “perfect” method, where he true edges are all ranked at the top will have an AUROC of 1. AUROC is widely used to summarize the content of a ROC curve because it has an intuitive meaning: the AUROC is the probability that if a positive and a negative edge are each picked at random from the list of edges, the positive will rank higher than the negative.

An alternative to ROC is the precision-recall curve. Precision, in the mathematics notation above, is the value , i.e., the number of true positives divided by the number of true positives plus false positives. Recall is the same as sensitivity, or true positive rate: it is . In other words, the PR curve contains the points (), as recall is usually plotted on the *x-*axis. The area under the precision-recall curve (AUPR) has an intuitive meaning just like AUROC. It is the average of precision across all recall values, or alternatively, the probability that if a “positive” edge is selected from the ranked list of the method, then an edge above it on the list will be “positive”. Neither precision-recall curves, nor AUPR are widely used. There is one problem with AUPR, which is that its value is dependent on the number of positive examples in the dataset. For this reason, it doesn’t make sense to average AUPR across datasets (while it does make sense for AUROC). For all of these reasons, I’m slightly uncomfortable with AUPR but that is not the main issue in the DREAM5 analysis. I have included an example of ROC and PR curves below. I generated them for the method “GENIE3” tested by Feizi *et al.*. This was the method with the best overall score. The figure below is for the *S. cerevisiae *dataset:

The ROC and a PR curves before (top) and after (bottom) applying network deconvolution to the GENIE3 network.

The red curve in the ROC plots is what one would see for a method without any predictive power (point #2 above). In this case, what the plot shows is that GENIE3 is effectively ranking the edges of the network randomly. The PR curve is showing that at all recall levels there is very little precision. **The difference between GENIE3 before and after network deconvolution is so small, that it is indistinguishable in the plots. **I had to create separate plots before and after network deconvolution because the curves literally overlapped and were not visible together. **The conclusion from plots such as these, should not be that there is statistically significance (in the difference between methods with/without network deconvolution, or in comparison to random), but rather that there is negligible effect.** There is a final ingredient that is needed to constitute “score”. Instead of just averaging AUROC and AUPR, both are first converted into *p*-values that measure the statistical significance of the method being different from random. The way this was done was to create random networks from the edges of the 35 methods, and then to measure their quality (by AUROC or AUPR) to obtain a distribution. The *p-*value for a given method was then taken to be the area under the probability density function to the right of the method’s value. The graph below shows the pdf for AUROC from the *S. cerevisae *DREAM5 data that was used by Feizi *et al. *to generate the scores:

Distribution of AUROC for random methods generated from the *S. cerevisiae *submissions in Marbach *et al.*

In other words, almost all random methods had an AUROC of around 0.51, so **any slight deviation from that was magnified in the computation of p-value, and then by taking the (negative) logarithm of that number a very high “score” was produced**. The scores were then taken to be the average of the AUROC and AUPR scores. I can understand why Feizi

*et al.*might be curious whether the difference between a method’s performance (before and after network deconvolution) is significantly different from random, but to replace magnitude of effect with statistical significance in this setting, with such small effect sizes, is to completely mask the fact that the methods are hardly distinguishable from random in the first place. To make concrete the implication of reporting the statistical significance instead of effect size, I examined the “significant” improvement of network deconvolution on the

*S. cerevisae*and other datasets when run with the protein parameters rather than the default (second figure above). Below I show the AUROC and AUPR plots for the dataset.

The Feizi *et al.* results before and after network deconvolution using alpha=1, beta=0.99 (shown with AUROC).

The Feizi *et al.* results before and after network deconvolution using alpha=1, beta=0.99 (shown with AUPR).

My conclusion was that **the use of “score” was basically a red herring**. **What looked like major differences between methods disappears into tiny effects in the experimental datasets, and even the in silico variations are greatly diminished**. The differences in AUROC of one part in 1000 hardly seem reasonable for concluding that network deconvolution works. Biologically, both results are that the methods cannot reliably predict edges in the network. With usable network deconvolution code at hand, I was curious about one final question. The main result of the DREAM5 paper

- D. Marbach
*et al.*, Wisdom of Crowds for Robust Gene Network Inference, Nature Methods 9 (2012), 796–804.

was that the community method was best. So I wondered whether network deconvolution would improve it. In particular, the community result shown in Feizi *et al. *was not a test of network deconvolution, it was simply a construction of the community from the 9 methods tested (two communities were constructed, one before and one after network deconvolution). To perform the test, I went to examine the DREAM5 data, available as supplementary material with the paper. I was extremely impressed with reproducibility. The participant submissions are all available, together with scripts that can be used to quickly obtain the results of the paper. However the data is not very usable. For example, what is provided is the top 100,000 edges that each method produced. But if one wants to use the full prediction of a method, it is not available. The implication of this in the context of network deconvolution is that it is not possible to test network deconvolution on the DREAM5 data without thresholding. Furthermore, in order to evaluate edges absolute value was applied to all the edge weights. Again, this makes the data much less useful for further experiments one may wish to conduct. In other words, DREAM5 is reproducible but not very usable. But since Feizi *et al.* suggest that network deconvolution can literally be run on anything with “indirect effect”, I decided to give it a spin. I did have to threshold the input (although fortunately, Feizi *et al.* have assured us that this is a fine way to run network deconvolution), so actually the experiment is entirely reasonable in terms of their paper. The figure is below (produced with the default network deconvolution parameters), but before looking at it, please accept my apology for making it. I really think its the most incoherent, illogical, meaningless and misleading figure I’ve ever made. But it does abide by the spirit of network deconvolution:

The DREAM5 results before and after network deconvolution.

Alas, **network deconvolution decreases the quality of the best method, namely the community method**.

**The wise crowds have been dumbed down.**In fact, 19/36 methods become worse, 4 stay the same, and only 13 improve. Moreover, network deconvolution decreases the quality of the top method in each dataset. The only methods with consistent improvements when network deconvolution is applied are the mutual information and correlation methods, poor performers overall, that Feizi

*et al.*ended up focusing on. I will acknowledge that one complaint (of the many possible) about my plot is that the overall results are dominated by the

*in silico*dataset. True- and I’ve tried to emphasize that by setting the y-axis to be the same in each dataset (unlike Feizi

*et al.*) But I think its clear that no matter how the datasets are combined into an overall score, the result is that network deconvolution is

**not**consistently improving methods. All of the analyses I’ve done were made possible thanks to the improved usability of network deconvolution. It is unfortunate that the result of the analyses is that network deconvolution should not be used. Still, I think this examples makes a good case for the fact that

**r**

**eproducibility is essential, but usability is more important.**

This is the third and final post in a series (part1, part2) of posts on two back-to-back papers published in Nature Biotechnology in August 2013:

- Baruch Barzel & Albert-László Barabási, Network link prediction by global silencing of indirect correlations,
*Nature Biotechnology***31**(8), 2013, p 720–725. doi:10.1038/nbt.2601 - Soheil Feizi, Daniel Marbach, Muriel Médard & Manolis Kellis, Network deconvolution as a general method to distinguish direct dependencies in networks,
*Nature Biotechnology***31**(8), 2013, p 726–733. doi:10.1038/nbt.2635

**An inconvenient request**

One of the great things about conferences is that there is time to chat in person with distant friends and collaborators. Last July, at the ISMB conference in Berlin, I found myself doing just that during one of the coffee breaks. Suddenly, Manolis Kellis approached me and asked to talk in private. The reason for his interruption: **he came to request that I remove an arXiv post** of mine, namely “Comment on ‘Evidence of Abundant and Purifying Selection in Humans for Recently Acquired Regulatory Functions“, a response to a paper by Ward and Kellis. Why? He pointed out that my arXiv post was ranking highly on Google. This was inconvenient for him, he explained, while insisting that it was wrong of me to post a criticism of his work on a forum where he could not directly respond. He suggested that it would be best to work out any issues I might have with his paper offline. Unfortunately, there was the inconvenient truth that arXiv postings cannot be removed. Unlike some journals, where, say, a supplement can be revised while having the original removed (see the figure switching of Feizi *et al.*), arXiv preprints are permanent.

My initial confusion quickly turned to anger. After all, my arXiv comment had been rejected from Science where I had submitted it as a technical comment on the Ward-Kellis paper. I had then put it on the arXiv as a last resort measure to at least have some record of my concerns publicly accessible. How is this wrong? Can one not critique the work of Manolis Kellis?

**Network nonsense begins**

My first review of a Manolis Kellis paper was in the fall of 2006, in my capacity as a program committee member of the * Research in Computational Molecular Biology* (RECOMB) conference held in Oakland, CA in 2007. Because Oakland is right next to Berkeley, a number of Berkeley professors were involved in organizing and running the conference. Terry Speed was chair of the program committee. I was out of the country that year on sabbatical at the University of Oxford, so I could not participate, or even attend, the conference, but I volunteered to serve on the program committee. For those not familiar with the RECOMB review process, it is modeled after the standard Computer Science conferences. The program committee chair forms the program committee, who are then assigned a handful of papers to review and score. Reviews are entered on a website, and after a brief period of online discussion about borderline papers, scores are revised and accept/reject decisions are made. Authors can revise their manuscripts, but the reviewers never see the papers again before publication in the proceedings.

One of the papers I was assigned to review was by a student named Joshua Grochow and his advisor Manolis Kellis. The paper was titled “Network Motif Discovery Using Subgraph Enumeration and Symmetry-Breaking“. Although networks were not my research focus at the time, and “symmetry-breaking” evoked in me nightmares from the physics underworld, I agreed to the review. The paper seemed to contain some interesting algorithms, appeared to have a combinatorial flavor, and potentially important applications- a good mix for RECOMB.

The problem addressed by Grochow & Kellis was that of identifying “network motifs” in biological networks. “Motifs” can be defined in a variety of ways, and the Grochow-Kellis objective was simple. In graph theoretic terms, given a graph *G*, the goal was to find subgraphs occurring with high multiplicity to an extent unlikely in a random graph. There are many models for random graphs, and the one that the results in Grochow-Kellis are based on is the Erdös-Renyi model (each edge chosen independently with some fixed probability). The reason this definition might be of biological interest, is that recurrent motifs interspersed in a graph are likely to represent evolutionarily conserved interaction modules.

The paper begins with a description of the method. I won’t go into the details here, except to say that everything seemed well until I read the caption of Figure 3. There the number 27,720 caught my eye.

During my first few years of graduate school I took many courses on enumerative and algebraic combinatorics. There are some numbers that combinatorialists just “know”. For example, seeing 42 emerge as the answer to a counting problem does not bring to mind Douglas Adams, but rather the vast literature on Catalan numbers and their connections to dozens of well-known counting problems. Similarly, a number such as 126 brings to mind binomial coefficients (), and with them the idea of counting the number of subsets of fixed size from a larger set. When I saw the number 27,720 I had a hunch that somehow some canonical combinatorial set had been enumerated. The idea may have entered my mind because of the picture of the “motif” in which I immediately recognized a clique (all vertices mutually connected) and a stable set (no pair of vertices connected). In any case, I realized that

.

The significance of this is that the “motif” on the left-hand side of Figure 3 had appeared many times because of a type of double- or rather thousandfold- counting. Instead of representing statistically significant recurring independent motifs, this “motif” arises because of a combinatorial artifact. In the specific example of Figure 3, the motif occurred once for any choice of 4 nodes from the clique of size 9, and any choice of 3 nodes from the stable set of size 12.

**The point is that in a graph, a ny**

**subgraph attached to a large clique (or stable set) will occur many times.**This simple observation is a result of the fact that there are many subgraphs of a clique (or stable set) that are identical. I realized that this meant that the Grochow-Kellis method was essentially a heuristic for finding cliques and stable sets in graphs. The particular “network motifs” they were pulling out were just subgraphs that happened to be connected to some large cliques and stable sets. There are two problems with this: first, a clique or a stable set can hardly be considered an interesting “network motif”. Moreover, the fact that they appear in biological networks much more than in Erdös-Renyi random graphs is not surprising. Second, there is a large literature on finding cliques in graphs, none of which Grochow-Kellis cited or seemed to be familiar with.

The question of the performance of the Grochow-Kellis algorithm is answered in their Figure 3 as well. There is a slightly larger motif consisting of **6 **nodes from the stable set of size 12, instead of 3. That motif occurs in all subsets of the stable set instead of subsets which means that **there is a motif that occurs 116,424 times**! **Grochow and Kellis’s algorithm did not even achieve its stated goal**. It really ought to have outputted the left hand side figure with six nodes in the stable set on the left, and not three. **In other words, this was a paper providing uninteresting solutions from a biological point of view, and doing so poorly to boot.**

I wrote up a detailed report on the paper, and posted it on the RECOMB review website together with poor scores reflecting my opinion that the paper *had *to be rejected. How could RECOMB, ostensibly the premier computer science conference on computational and algorithmic biology, publish a paper with neither a computational nor biological result? Not to mention an algorithm that demonstratably did *not* find the most frequently occurring motif.

As you might already guess, my rejection was subsequently overruled. I don’t know who made the final decision to ** accept** the Grochow & Kellis paper to the RECOMB conference, although presumably it was the program committee chair. The decision jarred with my sense of scientific integrity. I had put considerable effort into reviewing the paper and understanding it, and I felt that I had provided a compelling objective argument for why the paper was fundamentally flawed- the fact that the results were trivial (and incorrect!) was not a subjective statement. At this point I need to point out that the RECOMB conference is quite difficult to get into. The acceptance rate for papers in 2007, consistent with other years, was 21.8%. I knew this meant that even a single very negative review, especially one with a compelling argument against the paper, almost certainly would lead to rejection of the paper. So I couldn’t understand then, nor do I still understand now, on what basis the decision was made to accept the paper. This bothered me greatly, and after much deliberation I started boycotting the conference. Despite publishing five RECOMB papers from 2000 to 2006 and regularly attending the meeting during that time, the continued poor decisions and haphazard standards for papers selected have led me to not return in almost 8 years.

Grochow and Kellis obviously received my review and considered how to “deal with it”. They added a section titled “The role of combinatorial effects”, in which they explained the origins of the number 27,720 that they gleaned from my report, but then spun the bad news they had received as “resulting from combinatorial connectivity patterns prevalent in larger network structures.” They then added that “…this combinatorial clustering effect brings into question the current definition of network motif” and proposed that “additional statistics…might well be suited to identify larger meaningful networks.” This is a lot like someone claiming to discover a bacteria whose DNA is arsenic-based and upon being told by others that the “discovery” is incorrect – in fact, that very bacteria seeks out phosphorous – responding that this is “really helpful” and that it “raises lots of new interesting open questions” about how arsenate gets into cells. Chutzpah. When you discover your work is flawed, the correct response is to retract it.

I don’t think people read papers very carefully. Joshua Grochow went on to win the MIT Charles and Jennifer Johnson Outstanding M. Eng. Thesis Award for his RECOMB work on network motif discovery. [Added February 18: Grochow and Kellis have posted a reply here].

**The nature of man**

I have to admit that after the Grochow-Kellis paper I was a bit skeptical of Kellis’ work. Not because of the paper itself (everyone makes mistakes), but because of the way he responded to my review. So a year and a half ago, when Manolis Kellis published a paper in an area I care about and am involved in, I may have had a negative prior. The paper was Luke Ward and Manolis Kellis “Evidence for Abundant and Purifying Selection in Humans for Recently Acquired Regulatory Functions”, *Science *337 (2012) . Having been involved with the ENCODE pilot, where I contributed to the multiple alignment sub-project, I was curious what comparative genomics insights the full-scale $130 million dollar project revealed. The press releases accompanying the Ward-Kellis paper (e.g. The Nature of Man, The Economist) were suggesting that Ward and Kellis had figured out what makes a human a human; my curiosity was understandably piqued.

Ward and Kellis combined population genomic data from the 1000 Genomes Project with biochemical data from the ENCODE project to look for signatures of human constraint in regulatory elements. Their analysis was based on measuring three different proxies for constraint: SNP density, heterozygosity and derived allele frequency. To identify specific classes of regulatory regions under constraint, aggregated regions associated with specific gene ontology (GO) categories were tested for significance. Reading the paper I was amazed to discover they found precisely two categories: retinal cone cell development and nerve growth factor receptor signaling. It was only upon reading the supplement that I discovered that their tests had produced 53 other GO categories as well (Table S5).

Despite the fact that the listed categories were required to pass a false discovery rate (FDR) threshold for both the heterozygosity and derived allele frequency (DAF) measures, **it was statistically invalid for them to highlight any specific GO category. FDR control merely guarantees a low false discovery rate among the entries in the entire list**. Moreover, there was no obvious explanation for why categories such as chromatin binding (which had a smaller DAF than nerve growth) or protein binding (with the smallest p-value) appeared to be under purifying selection. As with the Feizi *et al. *paper, the supplement produced a story much less clean than the one presented in the main body of the paper. In fact, retinal cone cell development and nerve growth factor were **33 and 34** out of the 55 listed GO categories when sorted by the DAF p-value (42 and 54 when sorted by heterozygosity p-value). In other words, **the story being sold in the paper was based on blatant statistically invalid cherry picking.**

The other result of the paper was an estimate that in addition to the 5% of the human genome conserved across mammalian genomes, at least another 4% has been subject to lineage-specific constraint. This result was based on adding up the estimates of constrained nucleotides from their Table S6 (using the derived allele frequency measure). These were calculated using a statistic that was computed as follows: for each one of ten bins determined according to estimated background selection strength, and for every feature *F*, the average DAF value *D _{F}* was rescaled to

,

where *D _{CNDC}* and

*D*were the bin-specific average DAFs of conserved non-degenerate coding regions and non-conserved non-ENCODE regions respectively. One problem with the statistic is that the non-conserved regions contain nucleotides not conserved in all mammals, which is not the same as nucleotides not conserved in any mammals. The latter would have been needed in order to identify human specific constraint. Second, the statistic

_{NCNE}*PUC*was used as a proxy for the proportion under constraint even though, as defined, it could be less than zero or greater than one. Indeed, in Table S6 there were four values among the confidence intervals for the estimated proportions using DAF that included values less than 0% or above 100%:

_{F }**Ward and Kellis were therefore proposing that some features might have a negative number of nucleotides under constraint**. Moreover, while it is possible that after further rescaling *PUC _{F }*might have correlated with the true proportion of nucleotides under constraint, there was no argument provided in the paper.

**Thus, while Ward and Kellis**.

*claimed*to have estimated the proportion of nucleotides under constraint, they had only computed a statistic named “proportion under constraint”Nicolas Bray and I wrote up these points in a short technical comment and submitted it to the journal Science early in November 2012. The comment was summarily rejected with a curt reply by senior editor Laura Zahn stating that “relative to other Technical Comments we have recently received we feel that the scope and focus of your comment make it more suitable for the Online Comments facility at Science, rather than as a candidate for publication as a Technical Comment.” It is worth noting that Science did decide to publish another comment: Phil Green and Brent Ewing’s, “Comment on’Evidence of Abundant and Purifying Selection in Humans for Recently Acquired Regulatory Functions‘”, *Science *10 (2013). Green and Ewing’s comment is *biological *in nature. Their concern is that “… the polymorphism trends are primarily attributable to mutational variation and technical artifacts rather than selection.” Its fine that Science decided to host a debate on a biology question on its pages, but **how can one debate the interpretation of results from a method, when the method is fundamentally flawed to begin with? **After all, our problem with PUC was much deeper than a “technical flaw”.

We decided at the end to place the comment in the arXiv. After doing so, it became apparent that it had little impact. Indeed, I have never received any feedback about it from anyone. Apparently even this was too much for Manolis Kellis.

**Methods matter**

By the time I noticed the Feizi *et al.* paper in the journal Nature Biotechnology early in August 2013, my experiences reading Kellis’ papers had subtly altered the dynamic between myself and the printed word. Usually, when I read a paper and I don’t understand something, I assume the fault lies with me. I think most people are like this. But now, when the Feizi *et al.* paper started to not make sense, I didn’t presume the problem was with me. I tried hard to give the paper a fair reading, but after a few paragraphs the spell of the authors was already broken. And so it is that Nicolas Bray and I came to figure out what was really going on in Feizi *et al.*, a project that eventually led us to also look at Barzel-Barabási.

Speaking frankly, it was difficult work to write the blog posts about these articles. In addition to the time it took, it was exhausting and exasperating to discover the flaws, fallacies and frauds. Both Nick and I prefer to do research. But we felt a responsibility to spell out in detail what had happened here. Manolis Kellis is not just any scientist. He has, and continues to play leading roles in major consortium projects such as mod-ENCODE and ENCODE, and he has served on numerous advisory committees for the NHGRI. He is a member of the GCAT (Genomics, Computational Biology and Technology) study section until 2018. That any person would swap out a key figure in a published paper without publishing a correction, and without informing the editor is astonishing. That a person with great responsibility towards scientists is an abuser of science is unacceptable.

Manolis Kellis’ behavior is part of a systemic problem in computational biology. The cross-fertilization of ideas between mathematics, statistics, computer science and biology is both an opportunity and a danger. It is not hard to peddle incoherent math to biologists, many of whom are literally math phobic. For example, a number of responses I’ve received to the Feizi *et al.* blog post have started with comments such as

“I don’t have the expertise to judge the math, …”

Similarly, it isn’t hard to fool mathematicians into believing biological fables. Many mathematicians throughout the country were recently convinced by Jonathan Rothberg to donate samples of their DNA so that they might find out “what makes them a genius”. Such mathematicians, and their colleagues in computer science and statistics, take at face value statements such as “we have figured out what makes a human human”. In the midst of such confusion, it is easy for an enterprising “computational person” to take advantage of the situation, and Kellis has.

I believe the solution for this problem is for computational biologists to start taking themselves more seriously. Whether serving as reviewers for journals, as panel members for funding agencies, on hiring/tenure committees, or writing articles, all of us have to tone down the hype and pay closer attention to the science. There are many examples of what this means: a review of a math/stats containing paper cannot be a single paragraph long and based on a hunch, and similarly computational biologists shouldn’t claim, as have many of the authors of papers I’ve reviewed in these posts, pathways to cure disease and explanations for what makes humans human. Don’t fool the biologists. Don’t fool the computer scientists, statisticians, and mathematicians.

The possibilities for computational methods in biology *are *unlimited. The future is exciting, and there are possibilities for significant advances in areas ranging from molecular and evolutionary biology to medicine. But money, citations and fame cannot rule the day. The details of the #methodsmatter.

This is the second in a series of three posts (part1, part3) on two back-to-back papers published in Nature Biotechnology in August 2013:

- Baruch Barzel & Albert-László Barabási, Network link prediction by global silencing of indirect correlations,
*Nature Biotechnology***31**(8), 2013, p 720–725. doi:10.1038/nbt.2601 - Soheil Feizi, Daniel Marbach, Muriel Médard & Manolis Kellis, Network deconvolution as a general method to distinguish direct dependencies in networks,
*Nature Biotechnology***31**(8), 2013, p 726–733. doi:10.1038/nbt.2635

The Barzel & Barabási paper we discussed in yesterday’s blog post is embarrassing for its math, shoddy “validations” and lack of results. However of the two papers above it is arguably the “better” one. That is because the paper by Soheil Feizi, Daniel Marbach, Muriel Médard & Manolis Kellis, in addition to having similar types of problems to the Barzel & Barabási paper, is also **dishonest** and **fraudulent**. For reasons that I will explain in the third and final post in this series, we (Nicolas Bray and I) started looking at the Feizi *et al.* paper shortly after it was published early in August 2013. This is the story:

Feizi *et al.*‘s paper describes a method called *network deconvolution* that in their own words provides “…a systematic method for inferring the direct dependencies in a network, corresponding to true interactions, and removing the effects of transitive relationships that result from indirect effects.” They claim that the method is a “foundational graph theoretic tool” and that it “is widely applicable for computing dependencies in network science across diverse disciplines.” This high brow language makes network deconvolution sounds very impressive, but what *exactly *is the method?

Feizi *et al.* would like you to believe that the method is what is described in their Figure 1, part of which is shown below:

This is a model for networks represented as matrices. In this model an unknown matrix with eigenvalues between -1 and 1 is to be inferred from an observed matrix that is related to via

(1)

The matrix represents “direct effects” and the sum of its powers “indirect effects”. It is probably worth noting at this point that there is no particular reason to believe that effects will behave in this manner in any particular system of interest, but we return to this later. The eigenvalue assumption on is required for the specification of the model, because without it the infinite sum generating does not converge. An elementary manipulation of (1) shows that the matrix can be recovered from by the formula

. (2)

Unfortunately, many matrices cannot be decomposed as an infinite sum of powers of some matrix as in equation (1), so **equation (2) cannot be applied directly to arbitrary data matrices.** The only mention of this issue in the main text of the paper is the statement that “This [eigenvalue] assumption can be achieved for any matrix by scaling the observed input network by a function of the magnitude of its eigenvalues.” This is true but incoherent. There are an infinite number of scalings that will satisfy the assumption, and while **the authors claim in their FAQ **that “the effect of linear scaling on the input matrix is that … it does not have an effect” this **is obviously false **(also if it has no effect why do they do it?). For example, as the scaling goes to zero, converges to .

**What the authors have actually done with their seemingly innocuous aside is fundamentally change their model**: now instead of (1), is given by

. (3)

The problem with this model is that given there are an infinite number of solutions for and . Feizi *et al.*‘s approach to dealing with this is to introduce a scaling parameter that must be chosen *a priori*. **They do not even mention the existence of this parameter anywhere in the main text. Instead**, **they choose to make a false statement in the caption of Figure 1** when they write “When these assumptions hold, network deconvolution removes all indirect flow effects and infers all direct interactions and weights exactly.” Even when satisfies the eigenvalue constraint, once it is scaled before applying equation (2) the matrix has probability zero of being recovered.

In the video below, recorded at the Banff workshop on Statistical Data Integration Challenges in Computational Biology: Regulatory Networks and Personalized Medicine (August 11–16, 2013), you can see an explanation of network deconvolution by the corresponding author Kellis himself. The first part of the clip is worth watching just to see Kellis describe inverting a matrix as a challenge and then explain the wrong way to do it. But the main point is at the end (best viewed full screen with HD):

Manolis Kellis received his B.S., M.S. and Ph.D degrees in computer science and electrical engineering from MIT, so it is hard to believe that he really thinks that solving (2), which requires nothing more than a matrix inversion, must be accomplished via eigendecomposition. In fact, inverting a *2000 x 2000* matrix in R is 50% slower using that approach. What is going on is that Kellis is hiding the fact that computation of the eigenvalues is used in Feizi *et al.* in order to set the scaling parameter, i.e. that he is actually solving (3) and not (2). Indeed, there is no mention of scaling in the video except for the mysteriously appearing footnote in the lower left-hand corner of the slide starting at 0:36 that is flashed for 2 seconds. **Did you have time to think through all the implications of the footnote in 2 seconds, or were you fooled? **

While it does not appear in the main text, the key scaling parameter in network deconvolution is acknowledged in the supplementary material of the paper (published with the main paper online July 14, 2013). In supplementary Figure 4, Feizi *et al. *show the performance of network deconvolution with different scaling parameters () on the three different datasets examined in the paper:

In the words of the authors, the point of this figure is that “…choosing close to one (i.e., considering higher order indirect interactions) leads to the **best** performance in all considered network deconvolution applications.” However, while the supplement revealed the existence of , it did not disclose the values used for the results in the paper.

We inquired with the authors and were surprised to discover that while was used for the protein networks and for the co-authorship network, was used for the DREAM5 regulatory networks violating their own advice. What rationale could there be for such a different choice, especially one very far away from the apparently optimal choice “near 1”? We asked the authors, who initially replied that the parameter setting didn’t make a difference. We then asked why the parameter would be set differently if its value didn’t make a difference; we never got an answer to this question.

**Although it may be hard to believe, this is where the story gets even murkier.**

Perhaps as a result of our queries, the scaling parameters were publicly disclosed by Feizi *et al.* in a correction to the original supplement posted on the Nature Biotechnology website on August 26, 2013. The correction states “Clarification has been made to Supplementary Notes 1.1, 1.3, 1.6 and Supplementary Figure 4 about the practical implementation of network deconvolution and parameter selection for application to the examples used in the paper. ” But **Supplementary Figure 4 was not clarified, it was changed, a change not even acknowledged by the authors, let alone explained.** Below is Figure S4 from the updated supplement for comparison with the one from the original supplement shown above.

The revised figure is qualitatively and quantitatively different from its predecessor. **A key point of the paper was changed- the scaling parameter is now seen to not be optimal near 1, but rather dataset dependent **(this is reflected in a corresponding change in the text: in reference to choosing close to 1 “best performance” was changed to “high performance”). Specifically, the regulatory network dataset now has an optimal value of close to 0.5, perhaps providing an answer to our question above about why was used for that dataset alone. However that explanation directly implies that the authors knew the original figure was incorrect. We are left with two questions:

1. If the authors believed their original figure and their original statement that close to 1 “leads to the best performance in all considered network deconvolution applications”, then why did they use for one of the applications?

2.When and how was the July 16th Figure S4 made?

**Manolis Kellis declined to answer question 1 and by not acknowledging the figure change in the correction did not permit the readers of Nature Biotechnology to ask question 2. **

Clearly we have fallen a long way from the clean and canonical-seeming Figure 1. We wish we could say we have reached the bottom, but there is still a ways to go. **It turns out that “network deconvolution” is actually neither of the two models (2) and (3) above, a fact revealed only upon reading the code distributed by the authors. **Here is what is in the code:

- affinely map the entries of the matrix to lie between 0 and 1,
- set the diagonal of the matrix to 0,
- threshold the matrix, keeping only the largest fraction of entries,
- symmetrize the matrix,
- scale the matrix so that inferred in the next step will have maximum eigenvalue ,
- apply formula (2),
- affinely map between 0 and 1 again.

The parameter is the scaling parameter we’ve already discussed, but **there is a second parameter** . When the Feizi *et al.* paper was first published, the parameter appeared in the code with default value 1, and was literally not mentioned in the paper. When asked, the authors revealed that it also takes on different values in the different experiments in the paper. In particular, the regulatory networks DREAM5 experiments used . The only guidance for how to set , now provided with the new version of the supplement, is “In practice, the threshold chosen for visualization and subsequent analysis will depend on the specific application.”

It is hard to say what steps 1–7 actually do. While it is impossible to give a simple analytic formula for this procedure as a whole, using the Sherman-Morrison formula we found that when applied to a correlation matrix *C*, steps 1, 2, 4, 6 and 7 produce a matrix whose *ij*th entry is (up to an affine mapping) where and *m* is the minimum entry of *C*. Omitting step 1 results in *P _{ii }*, the inverse correlation matrix, so the effect of the mapping in this case is the addition of the final two terms whose possible meaning escapes us.

**We are left to wonder why the authors chose to add this step which lacks any apparent theoretical justification**. This bring us to question the meaning of the contribution of author Muriel Médard, a professor of information theory at MIT. According to the contributions section she is the one who “contributed to correctness proof and robustness analysis”. Presumably “correctness” meant describing the two steps needed to show that the solution to (1) is (2). But what would actually be relevant to the paper is a theorem about steps 1–7. Unfortunately such a theorem is hard to imagine.

When Feizi *et al.* was published in August 2013, they set up a companion website at http://compbio.mit.edu/nd/ where the code and data was made available (the data is no longer available at the site). On December 4, 2013, the authors updated the website. While there used to be one version of the program, there are now two different versions, one specifically for use on regulatory networks where the default values for the parameters are and the other with defaults . **“Network deconvolution” is supposedly a universal method applicable to any kind of network. Will the authors continue to distribute different domain-specific versions of the program, each with their own “default” parameters somehow divined by themselves?**

To summarize, “network deconvolution”, as best as we can currently tell, consists of steps 1–7 above with the parameters:

- DREAM5 challenge: and .
- Protein network: and .
- Co-authorship: and .

The description of the method in the main text of the paper is different from what is in the online methods, which in turn is different from what is in the supplement, which in turn is different from what is actually in each of the (two) released programs, namely the ad-hoc heuristic steps we have described above.

Having said all of that, one might be curious whether the “method” actually works in practice. Sometimes heuristics work well for reasons that are not well understood (although they are usually not published in journals such as Nature Biotechnology). Unfortunately, we have to disclose that we don’t know how the method performs on the datasets in the paper. We tried really hard to replicate the results of the paper, running the software on the provided input matrices and comparing them to the distributed output matrices (available on the NBT website). Despite our best attempts, and occasionally getting very close results, **we have been unable to replicate the results of the paper**. This bothers us to such an extent that **I, Lior Pachter, am hereby offering $100** to anyone (including Feizi *et al.*) who can provide me with the code and input to run network deconvolution and produce exactly the figures in the paper (including *both* versions of Figure S4) and the output matrices in the supplementary data.

Because we couldn’t replicate the results of the paper, we decided to examine how FK performs in one* *case for which Feizi *et al*. originally claimed (Supplement version July 14 2013) that their method is optimal. They stated “if the observed network is a covariance matrix of jointly Gaussian variables, ND infers direct interactions using global partial correlations”. This claim was deleted without mention in the updated supplement, however even if network deconvolution is not optimal for inferring a network from a correlation matrix (presumably what they meant in the original statement), the claimed universality suggested in the paper implies that it should be very good on *any *input.

The figure above shows a comparison of the Feizi* et al. *method (FK) with the various beta parameters used in their paper to regularized partial correlation, the “geometric root” (equation (2)) and the naïve method of simply selecting the top entries of the correlation matrix. In order to test the method we performed 40 simulations of sampling 500 observations from a random Gaussian graphical model with 1000 variables and an edge density of 5% to ensure the graph was connected yet sparse. Performance was assessed by comparing the ranking of the edges to the true edges present in the model and computing the area under the corresponding ROC curve. As a benchmark we applied partial correlation structural inference based on a James-Stein shrinkage estimation of the covariance matrix (a standard approach to gene regulatory network inference, described in Schäfer, J. & Strimmer, K. A Shrinkage Approach to Large-Scale Covariance Matrix Estimation and Implications for Functional Genomics. *Statistical Applications in Genetics and Molecular Biology* **4,** (2005)).

That FK does better than the most naïve method and yet worse than a method actually designed for this task is perhaps to be expected for a heuristic method that is based on a metaphor (one that dates back to Seawall Wright’s work on path coefficients in the 1920s- although Wright used (1) in the restricted setting of directed acyclic graphs where it makes sense). It is possible that in some contexts, FK will make things somewhat better. However what such contexts might be is unclear. The only guidance that Feizi *et al.* provide on the assumptions needed to run their method is that they state in the supplement that **the model assumes that networks are “l****inear time-invariant flow-preserving operators”**. While it is true that individual parts of that phrase mean something in certain contexts, the complete phrase is word salad. We wonder: is co-authorship flow preserving?

**To conclude: Feizi et al. have written a paper that appears to be about inference of edges in networks based on a theoretically justifiable model but**

**the method used to obtain the results in the paper is completely different than the idealized version sold in the main text of the paper and****the method actually used has parameters that need to be set, yet no approach to setting them is provided. Even worse,****the authors appear to have deliberately tried to hide the existence of the parameters. It looks like****the reason for covering up the existence of parameters is that the parameters were tuned to obtain the results.****Moreover,****the results are not reproducible. The provided data and software is not enough to replicate even a single figure in the paper. This is disturbing because****the performance of the method on the simplest of all examples, a correlation matrix arising from a Gaussian graphical model, is poor.**

**In academia the word fraudulent is usually reserved for outright forgery. However given what appears to be deliberate hiding, twisting and torturing of the facts by Feizi et al., we think that fraud (“deception deliberately practiced in order to secure unfair or unlawful gain”) is a reasonable characterization. If the paper had been presented in a straightforward manner, would it have been accepted by Nature Biotechnology?
**

*Post scriptum*. After their paper was published, Feizi *et al.* issued some press releases. They explain how original and amazing their work is. Kellis compliments himself by explaining that “Introducing such a foundational operation on networks seems surprising in this day and age” and Médard adds that “the tool can be applied to networks of arbitrary dimension.” They describe the method itself as a way to have “…expressed the matrix representing all element correlations as a function of its principal components, and corresponding weights for each component” even though principal components has *nothing* to do with anything in their paper. As with Barzel-Barabási, this seems worthy of nomination for the Pressies.

*Post post scriptum: *In September 2013, we submitted a short commentary to Nature Biotechnology with many of the points made above. While it was rejected four months later, we really *really *wish we could share the reviews here. Unfortunately they were sent to us with a confidentiality restriction, though having said that, any of the reviewers are hereby invited to guest post on network science on this blog. We were disappointed that Nature Biotechnology would not publish the commentary, but not surprised. Frankly, it would have been too much to expect them and their reviewers to unravel the layers of deception in Feizi *et al. *in the initial review process; it took us an extraordinary amount of time and effort. Having published the paper, and without a clear path to unpublication (retraction is usually reserved for faking of experimental data), there were presumably not many options. The question is whether anything will change. Feizi *et al. *apparently have more Nature Biotechnology papers on the way as evidenced by an extraordinary declaration on Soheil Feizi’s publication page (this is a pdf of the version when this blog was posted; Feizi changed his site an hour later) where the publication venue of their next paper appears to have been preordained:

S. Feizi, G. Quon , M. Mendoza, M. Medard, M. Kellis

*Comparative Analysis of Modular Integrative Regulatory Networks across Fly, Worm and Human*

`in preparation for Nature Biotechnology.`

The term “in preparation” is common on academic websites, especially on sites of graduate students or postdocs who are eager to advertise forthcoming work, but “in preparation **for**” really requires some hubris. We have to wonder, why do Feizi *et al. *assume that peer review is just a formality in the journal Nature Biotechnology?

[Addendum 2/23: The authors posted a rebuttal to this blog post, which we replied to here.]

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