The state of Influenza Phylogenetics

I really was not particularly interested in viral phylogenetics for most of my research career but I started my research in the area when I joined the Institute of Animal Health. I have a background in synthetic organic chemistry and protein crystallography and so I am well aware of how vicious and petty academic politics can be (ask me someday about the MRC skiing anecdote, or the Ribosome Nobel Prize story, or maybe the Rubisco saga or you can ask me about Nicolaou and Taxol ).

But I can honestly say that I have never met a more political, corrupt and inept field than influenza phylogenetics. It is staggering how bad it is, and that might be what makes it interesting. I come to it from a statistics perspective as I spent five years on my Damascene transformation in the statistics department at Oxford. I am very interested in bad science, people doing science badly in order to get grants and power but who really have no idea what they are doing. I was inspired by the work of John Ziman and the growth of the field of reproducibility (scientists like to suggest that this only applies to social sciences although psychology is acknowledging it has a problem too and biology in general definitely has a reproducibility problem). In viral phylogenetics there is a lot of bad science.

First I want to set out the main problems I have are:

1. Most biologists in the field have no idea what they are talking about from a theoretical perspective. They don't get the maths, they do not understand the assumptions and they ignore any results that do not fit with their expectations instead of asking themselves why it happened.
2. Sampling is terrible, It is all a convenience sample and this cannot avoid being a biased sample. Why are we focused on China when we collect almost nothing in Africa and the last swine flu pandemic came from Mexico?
3. People horde data and do not collaborate. There are 3 main databases which all have data in different formats with different annotations that you might or might not want. They are designed to make it difficult for researchers to use data from all three.
4. Peer review is intensely political and there are cliques which cite each other's papers excessively and which block publication of other groups. There are some government laboratories that have > 50 cites on a scientific note paper that says ... well not very much. Everyone is playing the citation game to keep their government laboratory funding. 
5. There is a lack of communication between the analysts and the laboratory scientists. For example how many analysts know that the virus was often passaged through hen's eggs for amplification before sequencing the result was that the sequences mutate to have chicken specific variations and so the sequences in the original sample are not the same as those they submit to the database. This is exactly the same as the cowpox vaccine for smallpox - it is NOT cowpox Jenner got lucky and also the problem of cell culture where the cell cultures are no longer the genotypes originally collected.

Why do I think these are problems?

1. I told a referee that phylogenetics is just a clustering method based on a metric. They assured me that this is not always true. For example parsimony and maximum likelihood are not distance based methods says the referee. Except fundamentally they are. To calculate a parsimony you need an alignment. To get a multiple alignment you need to build an alignment you use a progressive alignment based on a guide tree which will often use UPGMA a distance-based method. Distance is fundamental to progressive alignment. Parsimony itself depends on the smallest number of changes - a distance. The scoring models you use as evolutionary models for measuring changes and calculating likelihoods are metrics for finding distances. Probability is a metric in measure theory it is a distance. You could use information theory but the difference in information is again a metric and a distance. Whatever way you try and cut it phylogenetics depends on distance and a metric and groups based on those metrics. It is clustering it does use metrics and because it uses metrics it is not completely objective it is subjective and metric dependent. People who do phylogenetics would do well to read Kaufman and Rousseeuw to understand why clustering needs care and why metrics are very interesting (I have a story about one of the authors as well which makes me reluctant to suggest reading his book but it is foundational).
2. The BOOTSTRAP - I don't know where to start. Nobody who is a biologist has bothered to do some simple experiments to check what is does and what it means with real data. For example to know how many bootstraps you need to use run it on your data with 50, 100, 150 and 200 and see if it has converged - you get the same numbers each time. From my experience 100 is more than enough. All of these referees and experimentalists using 500 or 1000 or 10,000 are wasting their time. If you read Efron's book he says 100 is empirically often enough although, in theory, the number should be something like the square of the number of sequences. If they had read Efron's book they might grasp the issues. That means going beyond his simplified paper saying what the bootstrap is and definitely going beyond taking Felsenstein's word for it. There is some fantastic work on this by Susan Holmes who worked with Efron. This is really great stuff but under-read and poorly understood. So much so that she has moved on to other things.
3. The need to make everything quantitative. Biology is NOT always quantitative. If I see a clade in a tree and it is monophyletic to a geographical location I believe that tree. I do not need to put a number on it. I could work out by permutation test how likely it is to get a clade that is monophyletic to a location but given the sampling is convenience sampling that is not probably going to be meaningful.
4. Creating trees for distinct species should make sense. We know and I mentioned before that influenza undergoes rapid change to obtain host specific mutations when it is introduced to a new host, such as passaging it in chicken eggs. We know this experimentally for ferrets as well.  Why would a host specific tree be a bad idea? A referee and an editor thought so in my H9N2 work until a paper taking the same approach was published in Nature and then what I had done was Ok and they allowed my appeal after stalling publication for 18 months and two journals with the same editor in both.

How would you hack an election?

If I wanted to win an election how would I do it?

In areas where there is no voter id what I need first is a list of past voting records. I need to know who doesn't vote. I need this because I am going to vote for them and I don't want any of them turning up by accident. People who are on the register but cannot vote because they are dead or incapacitated look like great candidates as well BUT these are too easy to check so actually I want to avoid this or someone might detect my fraud quite easily.

Once I have a lost of non-voters who are on the roll and alive and who could vote but never do what do I do next? I go to the polling station on their behalf and vote for them. Remember that they never vote and they are likely not to be recognised. I need to do this in small numbers at all of the polling stations. The more stations the better to spread the load and hide what I am doing. I don't want to do more than 50-100 votes in each of the stations. This means that my voting team needs to be at a maximum of 100 people. I do not want to have the same person voting at a station twice. These teams then move from station to station over the day. I can also do postal votes and absentee votes and these help. This is a big logistical operation but I don't care if I win the prize and control the government. You are talking thousands of operatives but they will target only a few key areas which can be flipped. Only marginals matter.

This is certainly possible for the swing states like Pennsylvania, Wisconsin etc. if I have Russian man-power available but I would need a forward base and a long term plan to get the people in place over time. Combine this with analytics, targeted media a big fake news operation and you can win the election.

How would you recognise this in the results? Can it be detected? The only thing that you see is a higher than expected turnout, unexpected demographic shifts and a high turnout from previous non-voters. But all of this is plausible deniability. There is not way an audit can say that this fraud exists unless you corroborate all of the suspicious votes. This is the perfect undetectable hack. Oddly enough Trump and Brexit both depended on previous non-voters.

Is it just me or does seasonal flu not make sense?

The NHS are warning that the UK is going to have a bad flu season this winter because there has been a bad flu season in the southern hemisphere this year.

But maybe they got a bad season this year because we got a bad season last year. Surely if this bad season alternates between northern and southern hemispheres one affecting the other then once a bad season starts all seasons have to be bad after that.

What is more puzzling is how is their bad season going to become our bad season? If it is being spread by air travel then why didn't we have a bad summer flu season? Why does it only kick off in our winter after their winter and flu season is over. The idea was that we get more flu and colds in winter because we spend more time in doors passing it to one another in the winter. I agree in an agrarian society where behaviour changes with seasons but I spend as much time in my office in the summer as I do in the winter. That is apart from the summer vacation but if I went to New Zealand won't I bring back the flu?

A big factor in my job and exposure is schools. The new school term brings colds and flu. Now we have much shorter summer holidays for schools in the UK we should see longer flu seasons if this is part of the cause for flu being seasonal. Seasonal flu is reality but our models as to why it is seasonal don't fit very well. We need to get some better models as to why it is seasonal and how it spreads between hemispheres.

Humidity and temperature have been shown to have effects but I suspect that there are more factors to take into account and imagining how flu seasons spread between hemispheres is another factor to consider.

How not to develop analytic talent

I wrote a review of the book Developing Analytic Talent by Vincent Granville and gave it a good bashing. But I could not do it justice to the total incompetence. Vincent Granville PhD is a perfect example of a snake-oil salesman. He speaks about his papers, his experience and the investment he has attracted, he talks about his books but a quick Google of his name just turns up a website which he set-up and which engages in some shady practices, including him writing articles pretending to be other authors, especially women in order to make it appear more gender neutral.

The review could not capture the many gems within the book so here are some of his best bits of writing.

Compound metrics are to base metrics what molecules are to atoms. Just like as few as seven atoms (oxygen, hydrogen, helium, carbon, sodium, chlorine and sulfur) can produce trillions of trillions of molecules and chemical compounds (a challenge for analytical and computational chemists designing molecules to cure cancer), the same combinatorial explosion takes place as you move from base to compound metrics. 

p110

Very nice but Helium is a noble gas and does not form compounds on Earth although it might do in special environments.  His PhD is not in chemistry

Apparently he also thinks that an app for pricing in amusement parks would be a good venture

Increase prices  and find optimum prices. (Obviously, it must be higher than current prices due to the extremely large and dense crowds visiting these parks, creating huge waiting lines and other hazards everywhere - from the few restaurants and bathrooms to the attractions).

p105

Alternatively they could build more bathrooms and more restaurants and make even more money from the large crowds rather than reducing foot-fall as people go elsewhere. Who are richer the owners of WallMart or the owners of Tiffany's? 

This however is the best and saved for page 174

The number of variables is assumed to be high, and the independent variables are highly correlated.

What? Wait let me see what the definition of independent variables is. That would be whose variation does NOT depend on the variation of another. That would mean not correlated. This is more than a slight howler this is so elementary that you cannot believe a single thing the author says. He then goes on to do regression in Excel.

On page 189 he talks about the possibility of getting negative variances - this is impossible. On page 190 he talks about the variance being bounded by 1 as a maximum. This is nonsense even with normalised data the variance = 1 V is not <1 as="" he="" nbsp="" p="" states.="">

How not to design a questionnaire. Lord Ashcroft's dangerous political polls.

Lord Ashcroft has had a big impact on election polling in the UK. He has even had favourable mentions with Nate Silver on his blogs. But I have been taking a deeper look into his polls.

I have to admit to some political activism and I was previously a Liberal Democrat Councillor. I collected canvassing data and fed it into the party's own analysis program and it makes predictions about the state of your campaign. Most of the time it got it pretty close to right and I won by the amount I expected. That was small local scale analysis.

Lord Ashcroft is well known and a Tory donor and he has been running polls in both the UK and more recently in the US. He conducted a lot of focus groups around the Trump election and also the Brexit vote. What concerns me is not the polls and the data, but the way the polls are carried out. Most specifically the questions that are used.

I was reading a story on CapX by Robert Colville about the demographic disaster awaiting the Conservative Party and he used some very particular phrasing.

If people think that multiculturalism, immigration, the internet, the green revolution and feminism are forces for good, they will vote Labour/Democrat. If they think they are bad, they will vote Tory/Republican.

Now who exactly thinks in those terms? Oh yes, there is a force for good and I am behind it 100%. People just never say that. Oh that is a force for evil/bad, I am really against that. These phrases are straight from Lord Ashcroft's poll published in his book Hopes and Fears. Participants had to chose on a scale whether they thought that:

• Feminism
• The green movement
• The internet
• Multiculturalism
• Immigration

have made America better or worse.

I use these questions for my second year statistics class as examples of non-scientific questions. These are in fact sound-bites and propaganda disguised as poll questions. They provide a frame of reference to lead participants towards where the person asking the questions wants to take them. The questions are based on invoking feelings and reactions and not actually obtaining rational responses.

Take for example this more detailed question.

Thinking about the following changes in America over recent years, do you think they have made America better or worse?

More lesbian and gay couples raising children

How can lesbian women and gay men raising children have ANY effect WHATSOEVER on whether a country is better or worse? What does it mean for a country to be better or worse? If it means economically less successful then how do lesbian and gay couples raising children affect the economy? If it means the country has gotten worse because of a rise in crime then again how is this caused by lesbian and gay couples raising children? The responses to this question will be pure framing based on current personal experience. If your standard of living has declined recently then you will respond that America has become worse, but the relationship to lesbian and gay couples is forgotten in the question. I could have asked the same question about America getting better or worse and used "More Bush family members raising children" and I would get the same response.

This sort of question is nonsense. It is intended to bias and put words into the participant's mouths by limiting the spectrum of possible causes. The motivation behind these polls is generating easily digestible journalistic content. They can then summarise the poll by saying a majority of participants think that feminism or multiculturalism has made America/UK worse. These are leading questions and the poll is at best meaningless and at worst pure propaganda. Polls like this are designed to influence the opinions of participants and not to actually discover what their opinions are.

We need to think seriously about the impacts of polling carried out in this way, as for me it raises ethical concerns about how they are being carried out and how they are being used.

Bill Gates, VR and Influenza Vaccines

Bill Gates was shown around the NIH where they wanted to show him how VR helps to create better vaccines.

As I said in another Blogpost. I began my research career in structure based drug design.  Then I learned that you models are only as good as the data you feed in and so I moved down the pipeline, first to crystallography and now to sequence data analysis. I know the limitations but the NIH wants money and they are less likely to talk about them.

The limitation in influenza research is sampling. We simply do not collect the data properly. We have lots of data from China because that is where we think future outbreaks might come from, but the last Swine Flu pandemic originated in Mexico. There is not enough systematic global collection of data. This means that unexpected changes catch us unaware. Most of the times we do pick the right vaccine candidates but sometimes we get it wrong. VR will not help this.

What will help is the IOT. That provides an opportunity for massive data collection. The Cloud allows us to share data globally. If we can stop the national laboratories from hoarding data this would also be a big step forward. The WHO also needs to be reformed to remove some of the political players who are a barrier to sharing. Scientists are bad politicians. My dad worked on a funding committee and they had to move meetings to secret locations because the scientists were always trying to lobby then and bully them into decisions. In influenza research there is a ruling clique that wishes to restrict research participation. When you mention citizen science or data sharing they have a fit.

So what should we be looking at today?

1) Why is there a wide-spread breeding failure in the Celtic seas? Is this related to the death of marine mammals? Both of these sets of species are possible influenza sources/sinks and it might be a good idea to do some influenza viral screens to see if a new more pathogenic strain has evolved. If it is an influenza it is probably H7 or H3 and that it can affect mammals would be a concern.

2) H5N8 from Viet Nam is a new emerging highly pathogenic H5 containing lineage. We had an outbreak in Korea that spread to North America and also Europe but it does not seem to be able to persist in either Europe or North America (although for North America that is disputed). However the most recent European cases are not related to the Gochang and Buan Korean lineages, but to a Viet Nam sequence that again spread via Korea.

The key is vigilance, wider participation and thinking outside the box. The bird breeding might be nothing and unrelated but lets do a quick check. Improved data collection and screening is where we will make the break-throughs not in the VR lab.

My F1000 paper on reassortment in H5N8 - even open peer review has flaws

This is really irritating me as this is version 3 of the same story. It is not even a particularly interesting story except it is if you think deeply about it.

What I want to show is that H5N8 in the US is a subtype that has been produced by multiple events where an H5 containing virus reassorts with an N8 containing virus. To do this I constructed a tree for ALL the H5 sequences in the database and ALL the N8 sequences in the database.

If I am wrong then the H5N8 sequences from the US would cluster in one group and not be spread across the tree in many distinct clades. They would at least be close neighbours. Am I wrong? Nope they are spread all over both trees. In the H5 tree there are lots of neighbouring H5 sequences that have been sampled but they are from other subtypes. In the N8 tree they are also widely spread with sequences from many other subtypes in between.

So here is the referees comment https://f1000research.com/articles/5-2463/v1#referee-response-18901

I am completely incredulous about how this is a possible argument for rejection.

If the referees are right int their arguments then you CAN have clades in a tree that are polyphyletic for subtype without reassortment being the cause. I can simply get from H5N2 to H5N8 by spontaneous mutation of the N2 to the N8 form. I can make hundreds of base changes and insertions and deletions and this is more likely than a simple reassortment event.

Now let me imagine this is not their argument and that they will admit that reassortment does exist in the clades but that they are not convinced about the specific H5N8 reassortment events. They are suggesting that these occur in both the N8 and H5 tree which are two independent samples in the same pattern by chance and that there is a need for the internal gene trees to corroborate these events. I say in the paper very clearly that I cannot KNOW what the origins of the H5 and N8 are and I can just make suppositions about them, but I know that a reassortment event has definitely occurred. Otherwise what are the chances that the H5 and N8 genes both undergo substantial mutational changes between samplings of H5N8 and that the neighbours include large numbers of sequences from many other subtypes? If this is true then H5N8 sampling must have been carried out appallingly or in a very biased manner while we detect all the other subtypes with ease.

To be honest I have the internal gene trees as I just finished them for another paper about a different subject and oddly enough they show exactly what I said in the paper. I was 100% right. These two referees are 100% WRONG. In fact their arguments are so illogical and unsupported by evidence that I was surprised that they were brave enough to put their names to them.

So let me ignore the sneering way the review is written, because everyone has a time in their life when they thing they know it all and recent graduates tend to fall into this trap more often than not. I know that I was the same 20 years ago when I started my career.

Let me consider how they use rhetoric in order to create a straw-man to knock down by suggesting that the paper is about discovering reassortment - it is not. The title is very clear it says reassortment in H5N8 and then only part of the H5N8 tree - the US part is actually the main focus of the study. It is a paper about a specific example and the dangers of collecting data by subtype as this gives an incomplete picture of sequence evolution in a segmented virus that can undergo reassortment.

This is the point. If the segments can reassort then they can pass between multiple subtypes in their evolutionary pathway. This is not rocket science this is just suggesting it is better to consider this possibility in trees and sampling and not just carry out phylogenetic analysis by subtype.

This gives me with two options:
1) The referees are idiots.
2) The referees forgot to declare the conflict of interest in that they have a skewed view-point in order to protect their existing work. This paper starts to undermine the idea of monophyletic clades for subtype which underpins the WHO nomenclature system for H5 which Justin Bahl helps to manage.

I do not think the referees are idiots, but I do think the second point is true and that there are good reasons why Dr Bahl should have declared a real and prejudicial interest and NOT taken up the review. Nobody is objective about seeing their work undermined, ever. I also think this is a good reason for me to ban Dr Bahl and Joseph Hicks from EVER reviewing any of my papers and for editors to consider any review that they provide with suspicion.