We’re the LilBUBome team, sequencing the genome of LilBUB, to find the mutations that cause her polydactyly, osteopetrosis, and overwhelming cuteness, and learn about human diseases! AMA!

How much is sequenced? What sort of things have you learned? I'm very excited to see what you discover! I love Lil BUB and she's a dwarf with extra digits like me haha!!

We have only sequenced a genomic region called the ZRS (0.00023% of BUB's genome). But the sequencing of this small part has revealed that LilBUB has a specific mutation there which explains why she has polydactyly.

The cool thing is that this mutation is exactly the same that Ernest Hemingway´s cat had, suggesting that they are (distant) relatives!!!

Hi,

we've only sequenced a tiny region so far and learned that BUB must be a distant relative of Ernest Hemingways cats (he had a number of cats with extra fingers) because both of them share the same mutation. - daniel (answer above is dario)

You can't assume that BUB and the Hemingway cats are related (beyond the obvious level of relatedness between all domesticated cats) just because they have the same mutation. You need to show that the mutation resides on a shared haplotype! Otherwise, it could easily be a recurrent mutation.

Yes, you are right, to prove the relatedness we would need to look at the shared haplotype. We can, however, assume that they might be related: our interpretation was primarily based on the original paper describing the Hemingway mutation, where it was shown that polydactyl cats on the East coast all share the same mutation and that this is different from the mutations found in the UK. But even beyond the question of relatedness, it's possible that BUB has an additional mutation somewhere, which amplifies the polydactyl phenotype. She has additional digits on ALL paws, whereas Hemingway cats are typically only polydactyl on 2. We hope we can answer this with the whole genome sequencing.

does the space planet that LilBub is from approve of your science?

we are the secret infiltrators from that planet trying to make you accustomed to our procedures and traditions. but don't tell anyone

What are you going to do with all of the data once you've finished? Are you planning to sequence any more special cats or animals?

You're doing such a cool thing!!

Thanks so much - we're glad you like the project. We're not planning to sequence any more special animals ourselves. LilBUB is really a labour of love for us, and we'd find it difficult to find another project that's quite so amazing. We're planning to share the data, though. This way other scientists can also access the information and maybe find stuff we missed. - uschi

Thanks!!! We would really like to share the data with the world so people can also have a look at it and help us to find the mutation(s). Any relevant finding will be of course reported on our blog as soon as we find it and eventually published as "open access"

After all, this project has been built with the help of the people so it must be accessible to everyone.

If we get more money than initially thought, we would like to use exceedings to help the 99catlives initiative headed by Prof. Leslie Lyons from the University of Michigan. Here we would sequence cats with with Hypertrophic Cardiomyopathy, a inherited disease in several cat breeds.

Does it take special/different equipment to sequence animals vs. people, or is it all the same?

It's all the same. Whatever type of DNA you put into the machine, it'll sequence it.

@shuttup_meg & tesformes you can see the actual machines that we'll use to sequence BUB in the video (second half https://experiment.com/lilbub)

Hey there! As an undergrad in biotech I really lightened up when I saw this AmA. My question is, are you planning on sequencing the whole genome ( I don't really think so, that would take a lot of time right?) or are you targeting specific areas?

If it's the latter I would like to know how you choose the specific areas you are about to sequence. Are those later compared with ones from "normal" cats?

Thanks in advance!

Hi, we are sequencing the whole genome. The process including the library preparation and sequencing takes a couple of weeks. So it is not that much time!! ;-)

1. Is Osteopetrosis a bad thing? Does it cause pain, or does it just make Lil Bub more durable over time?

2. Could Lil Bub's "cute" traits be isolated and bred into other cats one day, without the corresponding health problems?

3. How much of sequencing DNA is automated, vs a manual process? How long will sequencing his genome take?

1. Yes, osteopetrosis CAN potentially be very bad. However, in BUBs case, with the treatment she got, it seems she's doing fine. For the human condition you can find a lot of information on the website of osteoporosis.org. One of the things we're interested in is how diverse osteopetrosis in cats, and whether all cats develop the same symptoms. (So far, it seems this is not the case)
2. For now what we can almost say for sure, is that the polydactyly and the osteopetrosis are probably caused by two separate mutations. This likely also means that through breeding she could pass on the extra digits, but not the osteopetrosis. However, because she is so small she really shouldn't be having kittens, it could be very harmful for her.

Sorry, autocorrect decided to change osteopetrosis.org into osteoporosis.org. The correct link is osteopetrosis.org (in the 1st answer). - uschi

Is a cat with osteopetrosis less susceptible to injury?

No, unfortunately not. In fact, with osteopetrosis bones become more brittle and are MORE likely to fracture.

How was Lil Bub treated for it? Wikipedia mentions bone marrow transplants and corticosteroids as treatments.

No, actually, Mike used a rather unusual therapy. But then again, there are many different types of osteopetrosis. When we asked our specialist on bone disorders about BUB's osteopetrosis and it seems it's not quite clear which type she has (see the interview here: https://experiment.com/u/JGWzkw ). Mike actually tried many different treatments, and they didn't really work. Then he tried something called Pulsed Electromagnetic Field Therapy, which has worked for BUB. We're not sure if this will also work for other cats with osteopetrosis, though.

1. The machines that do the DNA-sequencing do this by themselves once you start the process. BUT before you can start the process you have to prepare the DNA (which is called Library-Preparation). This can can take a few days. Then, once the machine starts sequencing, the whole genome will be sequenced in about a week.

What type of machines are you using for your sequencing and/or library preparation?

We'll be sequencing the genome on Illumina HiSeq2500 machines. Do you know about them or do you want to know a little bit more?

I know a little about sequencing, but am essentially a beginner (with an electrical eng background, not biochemistry), so yeah I'd love to hear more detail about the mechanics of your work.

Actually is a really cool system!!!

There is a really nice explanation and video here:

http://www.illumina.com/systems/hiseq_2500_1500/technology.html

Hi to all the illumina personnel who are reading this!

:) but are they? ähem, we could also use a SOLiD, a Proton, a Nanopore or a 454. We are amazed about the technique, but much more we're looking forward to the genome sequence.

What is your favorite thing about Lil BUB? :)

Sounds cheesy, but her spirit. She seems to spread good vibes everywhere she looks. We contacted her and her dude with our idea and it's been a really positive experience so far.

Do you get to cuddle her when you're not doing experiments? It must be scary to the little sweetie.

Hey, unfortunately no. In fact we've never met LilBUB "in person". And luckily sequencing her genome does not hurt her at all - we got a blood sample (taken by her vet, during her regular check-up). We'd love to meet BUB though! Maybe some time in the future...

and I for one get to cuddle my cat Castro when i'm not doing experiments. He seems to enjoy it (and then he walks away) - daniel

Do you think that there might be human patients that suffer from Lilbub's disorder?

we don't know but we think that could be possible. there are some patients that have very similar looking bones.

the question you asked is actually one of the reasons why we want to find out about BUB -daniel

what sequencer do you use?

are you going to enrich the dna during library prep?

Hi, we use Illumina HiSeq2500 sequencers. Since we want to do whole genome sequencing there'll be no enrichment during Library-Prep just a few PCR-cycles. and we'll be sequencing the genome as paired-end 100bp to get a 40X coverage. - daniel

Can you confirm that no BUBs were harmed by these experiments?

confirmed. not at all

Yay! GOOD JOB, LILBUBOME!

Thanks!!

How cute do you think lil bub is on a scale of 1-10?

11

at least

Did any of BUB's siblings/ parents have any of her traits, were they genetic? Or are these special traits "accidental"?

Nice question!! We do not have much information about her relatives but she was found as the runt of a healthy feral litter.

We know now that the polydactyly and the osteopetrosis come from different mutations. For the polydactyly we think that at least one of her parents should be affected by that, because the Hemingway mutation that BUB has is not entirely uncommon in american cats. Cats with this type of polydactyly resulting from this mutation are completely healthy besides this.

For the osteopetrosis, we think that is probably an accident as this disease may affect their reproductive capabilities

My understanding is that BUB is a rescue cat, and therefore no one knows who her siblings or parents are - otherwise they'd be able to test them, too.

oh, shoot! a litter of BUBs would be the best

The only choice we have is to accelerate cloning technology.

A world of BUB.

We all love BUB, but in the remote case that there would be that possibility, we definitely dont want to clone her!! That would origin an animal that would suffer from osteopetrosis.

Lets just enjoy her alone. After,all, she is one of a kind ;-)

Do you think with all of her problems, it is more likely that she has a ploidy problem, a translocation or a microdeletion affecting a chromosomal region rather than a series of individual genetic mutations? Are you going to try doing some karyotyping or cytogenetics before you start the sequencing to give you a better idea of this? How much is known about aneu/polyploidy in cats?

Great question. We're actually not sure how much is known about aneuploidy in cats. But we're hoping that when we do whole genome sequencing, with the right analysis we'll also find out about large chromosomal abnormalities. Daniel and Darios group also has a lot of exertise for finding smaller structural variants that can cause disease in patients. -uschi

You think in the future it will be possible to make someone cute?

Cuteness is in the eye of the beholder - now&in the future. Except if you're LilBUB, in which case cuteness is a state of being.

So their isn't a chance to make me cute? I just want her to like me :(

Hmm, as basic researchers we are a bit out of our depth to answer this.

Why did you decide to do this, and what can be done with the results?

This is the first AMA I've been around for, lol.

It's our first AMA, too :) We decided to do the LilBUBome for multiple reasons: First of all, LilBUB is great, and we were really, really curious if we could apply our expertise in genetics to better understand her "magic". And then we realised that if we did this as a crowdfunding project we could engage people, and tell them about (our) science and rare diseases. For us, this is really important. Anyone can understand genetics, as long as it's explained well.

What could the results be good for? Initially it's all about the knowledge. BUB has a rare disease that is obviously very unique, similar to patients with other rare diseases. For these patients it's often very important to know the exact cause of their disease. For example for optimised treatment, or for telling if future siblings or kids might also be affected. Also, because theses cases are so rare, it's helpful if they can be compared to other, similar cases. Does that make some sense to you?

How did you get involved with Lil Bub?

We saw the documentary on vice about BUB and were totally fascinated. And then, because we're geneticists, thought we would really like to try to find her mutation.

Then we sat down and wrote her dude an email suggesting that. and guess what, he was up for it!

Are you planning to do a MicroArray as well?

No, we're hoping that whole genome sequencing will give us all the information we need. - uschi

Have you considered trying to pinpoint a possible genetic cause for Henri's ennui?

No, sorry. We just find BUB slightly more......happy ;-)

Once you have sequenced the lil'bubome, and isolated the cute gene;

do you plan to weaponize it?

Well, this is quite far away from our goal. And we do not like weapons thath much.

But it would be great if conflicts could be solved with cuteness instead of weapons. Everything would be better ;-)

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Also -- congrats on bringing science to a new audience with this. I love cats and science, so as soon as I saw this I had to back it. I have my fingers crossed you'll reach your goal!

thank you so much. we're having a great time with this and I hope we can make science a little bit less mysterious in the process!

Hello, I was wondering apart from the polydactyly and osteopetrosis and the mutation that causes the under developed jaw and lack of teeth, what else are your expecting to find from Bub's genome? I'm not sure if its just me or what, but her eyes look exceptionally huge to me, do you think there's a genetic basis to that? What do you aim in sequencing Bub's genome? I'm sorry for the many question!

it's hard to say wether her eyes and maybe also her tongue are larger than normal. I talked to Dr. Woodruf about it and he doesn't think so. It might be that BUBs head is a little bit smaller - which could be also explained by her bone overgrowth - and this is why her eyes/tongue appear larger

that being said: for the case that we find the causative mutation in a specific gene (for example), we can look at what is known about that gene and then see wether it is not only involved in bone growth, but maybe also in eye development for example. but that will only be possible if we find the mutation

How is it that Bub's head is smaller when she is experiencing an increase in bone density? Should not her head be larger instead? Please do explain :)

Smart question ;-)

What happened is that osteopetrosis was affecting her during development. For the proper bone to develop, first there is a cartilage template that is formed and then it mineralizes.

In this case the mineralization probably happened too fast before the complete cartilage template was formed and this is why she has a reduced skull

I love geneticists! I love LILBUB!! Therefore I love this AMA!! (even though I'm late). I live near another Max Planck Institute - what got both of you into genetics, especially cat genetics (best imo)?

Hi. I can only speak for myself, of course. I studied Biology, did all the normal stuff (Birds, Bees, and Botany) and then I had a seminar about developmental genetics. Fruit flies, segmentation, gap, and Hox genes. When I heard that the first time I thought this was the absolutely coolest thing I have ever seen. And from there I went more and more into developmental genetics and gene regulation. And because the genetic mechanisms are basically the same between all animals and there are so many amazing species that aren't studied I have a soft spot for unusual cases. - daniel

Would you consider sequencing the genome of grumpy cat one day? :)

Not really!! This project makes sense to us because there is a scientific reason behind. There is a mutation associated to a disease (osteopetrosis) that is related to our scientific background and that we would like to study and understand the cause.

Although grumpy cat has dwarfism and might be interesting to study, we prefer to focus on LilBUB. We really love her and want to know more about her!!

How do you differentiate between 'junk DNA' and coding DNA, when selecting fragments to sequence if you can only partially sequence a genome?

If you wish to sequence only a part of the genome, lets say all the coding DNA you can enrich for it. To enrich, you 1) isolate all genomic DNA 2) break it up into little pieces (each ca. 300bp long) 3) fish out all of the regions matching to the coding sequence and 4) then sequence only the fished out pieces of DNA. Brief enough or more detail?

Hey, great work you're doing here so far. Could you go a bit into the general process of how genome sequencing is done, and knowing what to look for in terms of mutations?

well, there are a number of different techniques, but the one that's the moste widespread at the moment is based on a technology that's called "sequencing by synthesis". In essence, you break the genome into millions of random, short pieces of DNA (200-300bp) and sequence all of them in parallel. Then you take all of these 200-300bp long fragments and puzzle them together by mapping them to a reference genome (if that exists). you do this so much, until you have every position in the genome covered 40times. once you're at this point you try to filter out all of the positions where the DNA you sequenced DIFFERS from the reference genome. These differences are mutations and everyone has thousands of little differences. You then try to filter and evaluate the mutations for ones that you think could be more or less likely disease-causing.

Since you're currently sequencing the genome, I assume there's no reference to compare it to? Or would you compare it to another of the same breed?

there is a reference to compare to, but this is from an Abasynnian (called Cinnamon).

Luckily, Leslie Lyons and her team, which we are collaborating with have started to sequence various cat genomes, so that we will have several references to compare to. check out her 99lives project: https://www.facebook.com/Feline99Lives

this will allow us to filter out mutations that BUB shares with other cats and that are likely benign

in BUB's case, the first thing we'll do is to look for mutations in genes that we know to cause osteoporosis in humans

Are you hiring? Quite serious. I'm an animal science major and graduating soon! This is amazing. Genetics and cats - my favourite project!

Hey, sorry but we're not hiring. We are doing the LilBUBome as a side project ourselves, beside our regular work. And it's definitely something out of the ordinary for us, too. But we hope you find something! Science is fun :)

I'm glad you're doing it! Thank you for answering!

Thanks!! :) Yeah, sorry for the late response. It was the middle of the night in Europe, and we all have day jobs...

Why is polydatyly in LilBUB such a focus when breeds like Maine Coons are known to commonly have the trait? Is it just the curiosity of that trait in conjunction with the osteopetrosis?

(Grew up with a lovely Maine Coon, polydactylic on every paw though he couldn't retract the 6th digit)

Well, initially we were drawn to it, because before we found the Hemingway mutation (we've written about it here: https://experiment.com/u/8jUcsg ) it wasn't at all clear if the polydactyly and the osteopetrosis were 2 separatae things, or if BUB had a special kind of mutation, which caused osteopetrosis AND polydactyly. Based on our data, it now seems it's the first case (2 separate mutations). On a more personal note, I am intrigued by all cases of polydactyly, because I did my PhD studying HOW mutations that cause polydactyly do so - like, the mechanistic basis of it. So, naturally, I like Maine Coons with their frequent polydactyly (well, also without :). In fact, it's rumored that the first Hemingway cat was a Maine Coone (or related to one?), though I'm not sure what the source of that rumor is, except that they frequently have polydactyly. - uschi

Will the mapping of a mutated cat's genome have real world application to human genetic research? Why not just sequence a humans genome? The whole sequencing while inexpensive sounds like a vanity project to me.

Well, it's basic research (trying to understand the world around us) with possible implications for human disease. What cat data can definitely add to human data is to really get a grasp of the fundamental underlying mechanisms. Using the evolutionary interconnectedness of mammals and shared developmental processes, we hope LilBUBs data will add insight into the common origins of cat and human phenotypes. We don't think it's a vanity project, but if it is we'll definitely aim for one next time that's less work ;)

A vast majority of scientific applications to human genetic research come from research and study of other organisms.

In the case of osteopetrosis, this disease has been described in different animals and humans and the genes responsible to some of its different variants (when found) seem to be conserved between species.

The specific combination of sympthoms that BUB showed has been never reported before. This led us to think that it could be a new variant of osteopetrosis caused by an unknown gene.

If we find the cause, this of course will relevant for cat health and treatment but might also be extended to other animals and humans.

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Is the cat retarded?

No, definitely not. BUB seems like a perfectly normal, happy cat.