Starting the New Year with some LilBUBome updates

Hi All,

Happy New Year! We hope everyone has had a great start to 2016!

We know things have been a bit quiet around the LilBUBome, but the second half of last year was a busy time for us: Daniel, Dario and the sequencing team at the Max Planck Institute completed the sequencing of BUB’s DNA (we blogged about it here and here), and we’ve since been analyzing the data and following up on candidate mutations. We also did a bit of outreach, talking at various venues about sequencing BUB’s genome with the help of crowdfunding – and what an amazing experience has been to work with such a special cat and so many supporters!

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In October we talked about the LilBUBome at the Sanger Institute postdoc retreat. While we were there we were allowed to have a quick look at their sequencing facility – and of course we took a picture for you!.

In addition, we’ve compiled a series of videos, which we’ll be releasing over the following weeks, where we try to explain what we did, why and how we did it and how you can go about sequencing a genome.

Today, let us introduce the first installment of the LilBUBome videolog. We hope you enjoy!

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Sequencing the LilBUBome: choosing the “right” technology…

…and the technical parameters of our sequencing run.

Last week we gave you a brief and general overview of the different technologies that are available if you want to sequence a genome. So, how did we end up selecting Illumina sequencing? Since we’re frequently asked about our choice of technology, this post is dedicated to this topic. We’ll also discuss some of the technical details of our sequencing run, so if you’re interested in these kinds of things, keep reading.

Questions about the LilBUBome sequencing technology during our Reddit AMA.

Basically, our choice was an absolutely pragmatic one: We wanted to maximize our chances of finding something. Now, in terms of sequencing probably any of the available technologies would have given us data, but choosing the right one can make a difference when it comes to analyze certain parts of the genome. We decided to work with a method that was well-established in the institute where Daniel and Darío work. Their institute has an excellent sequencing facility, which routinely does Illumina sequencing and lots of complicated analysis, and so we have relied on their help and expertise throughout the hands-on part of the LilBUBome. Hence, why we chose Illumina.

As an added bonus there have been some unexpected benefits:

  1. As you may know, we are doing the LilBUBome as a labour of love, in addition to our regular work. We did not expect, however, that collaborating scientists would do the same. But they did! So we would like to use this opportunity to thank the staff of the Max Planck Institute’s Sequencing Facility, who have generously donated their hands and minds to the LilBUBome, completely free of charge! You already know Norbert from previous posts (for example here and here), but the same acknowledgment can be extended to the whole team. You are great!!!

    seqcore

    Back row: Norbert Mages, Myriam Hochradel, Heiner Kuhl; Middle row: Daniela Roth, Sven Klages, Sonia Paturej, Bernd Timmermann; Front row: Hannes Cash, Ilona Hauenschild, Stefan Börno; Martin Kerick is missing in the pic, but we also love you 😉

  2. After we had completed crowdfunding and were preparing for the actual sequencing, Illumina kindly offered to provide us with free reagents for the sequencing. After some hesitation, we decided to accept the offer. Primarily, because it allowed us to upgrade the technology* – we were able to sequence Lil BUBs genome in more depth and quality, thus increasing our chances of success. In addition, we could use a method and a machine that’s much faster than the one we originally wanted to use (20 hours compared to 11 days!!!). And finally, this also means that we can make your donations go even further, to fully verify our findings from the sequencing (stay tuned!) with additional experiments.

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* And finally, here – as promised – the Technical details (aka TechTalk):
Sample preparation kit: Truseq sample prep for Next Seq Version2. Version 2 gives slightly longer reads and the coverage is much more even than with Version 1.
DNA fragmentation: We used a Covaris machine, according to the standard protocol provided by Illumina. We made 2 libraries (300bp and 500bp length) and then ligated adaptors. After adaptor ligation (Adaptors were roughly 120bp each), the libraries were run on a gel and the bands (sized 400-500bp for the 300bp library and 600-700bp for the 500bp library) were excised from the gel.
Seq machine: NextSeq 500. (Before the Illumina donation we would have used the HiSeq 2500. The NextSeq gave longer reads, and also the sequencing protocol was much faster (20 hours on the NextSeq vs 10-11 days of the Hiseq))
Read lenght : 2x 150bp. (If we would have used the HiSeq, we would have sequenced 2x 125bp reads. The longer reads from the NextSeq don’t improve coverage (40X), but they do allow for better assembly. Better assembly is crucial to detect small rearrangements.)
Paired end: Yes

The technology behind the LilBUBome: sequencing

Since we’ll be talking a lot about sequencing and sequencing results in the next few weeks, we’d like to start by telling you about the technology we used.

Generally speaking if you want to sequence something today there are two approaches: Sanger sequencing or next-generation sequencing (NGS) methods. NGS methods are the go-to methods if you want to sequence a complete genome (as we did with LilBUB), because the cost to sequence a single base (the building blocks of DNA) is much less and you can work at high-throughput (sequence a lot of DNA at the same time). Having said that it’s still a couple of thousand dollars per genome, so Sanger sequencing may be a better choice if you’re only interested in sequencing a small segment of DNA. Previously, we used Sanger sequencing to find the mutation that’s responsible for BUB’s polydactyly.

This time, to sequence the whole genome of Lil BUB we used NGS, though. More specifically, we used Illumina sequencing. Continue reading

Same, same, but different? Meet Rosie, another cat with osteopetrosis.

One of the great things about the LilBUBome is that it’s an open science project, so we get to interact a lot with people who are interested in our science. One of these people is Ann, whom we got to know through Mike (BUB’s dude). Ann had contacted Mike a few months ago, because she also has a cat, Rosie, who was recently diagnosed with osteopetrosis, and Ann wanted to know more about the disease.

Of course, we were really fascinated by Ann & Rosie’s story: osteopetrosis is a rare disease, and even more rare in cats. BUB seems unique, because she’s the only cat with a juvenile (early-onset) form of the disease. Rosie’s case seems different.

Rosie & Ziggy April 2015

A furball with piercing green eyes. Meet Rosie, another cat with osteopetrosis.

For example, she only developed osteopetrosis when she was older, so – unlike BUB –  she’s actually quite long and lean (BUB is probably so small because her osteopetrosis forced her bones to calcify and stop growing too quickly).

Also, Rosie has different symptoms than BUB, and also received a different treatment. However, similarly to BUB she seems to be doing fine, despite living with osteopetrosis.

So, similarly to human patients with osteopetrosis, the disease seems to be rather diverse in cats, too. To get a better picture, we asked Anne to tell us Rosie’s story. Continue reading

How to find a mutation? – Needles and haystacks

Finding a disease-causing mutation is like finding a needle in a haystack. Or rather like finding one needle in hundreds of haystacks. As in: it’s really difficult. Because the genome is a BIG place, and you’re looking for one small change*. So how do you go about finding such a change?

Finding a mutation is like finding a needle in a haystack (picture by t_buchtele via flickr).

Finding a mutation is like finding a needle in a haystack (picture by t_buchtele via flickr).

Basically, there are two strategies: Continue reading

How to make a LilBubome – Step 1: Extract DNA

You might have already seen it in the video or in our last lab note: We recently received a blood sample from LilBUB. The blood was taken from her during her routine medical check-up with her vet, Dr. Woodruff.

And now that the sample has arrived in our lab, we can start the actual work! For this, what we need to do first, is to extract the DNA from the blood sample. Might sounds sophisticated, but actually: not at all!!!!

DNA extraction is composed by 2 main steps: Continue reading

Why sequence Lil Bub? – 1. For better diagnosis and treatment

Many people have asked us why we want to sequence Lil Bub? To satisfy our scientific ego? Because we can? Well, curiosity is part of it, but there’s much more! First and foremost, because we think it can help pets and humans with traits similar to Lil Bub if we can find the genetic cause(s) of Lil Bub’s special looks. It could help diagnosis and also guide treatment(s).

Lil Bub’s osteopetrosis
Lil Bub has a very rare bone disorder, which has been diagnosed as osteopetrosis. It means that her bones become more dense with age, and when she was about one year old, this bone disease was so painful that she could barely move. Luckily Mike, her owner, was very persistent, and with the advice of fans and specialists, they found a treatment that worked for Lil Bub. It’s called Pulsed Electromagnetic Field Therapy. This is a video about Lil Bub’s miraculous recovery:

Does this mean that her therapy is a good treatment for other cats with osteopetrosis? Does this mean that people with osteopetrosis should also be treated with radiotherapy? Is cat osteopetrosis the same as human osteopetrosis? Currently, the answer is – we don’t know. But if you know what causes the osteopetrosis you can start answering these questions. Continue reading

From genetics to traits – Screentime

Last week, we posted some background info about the scientific principles behind the LilBubome. Specifically, we introduced the concept of DNA, and that it is the unique combination of DNA building blocks, which gives everyone their individual traits. Importantly, these principles are universal across all walks of life. If you’re interested in the topic, and especially the relevance for human traits and diseases, here’s a two-hour show from PBS from 2001, when the human genome was first sequenced. It explains about the things you can read out of a genome sequence – we’ll recap this, when we explain about the things we are looking for in Lil Bub’s genome. The show is great, but please note that there are some inaccuracies. In particular, the sequencing technology they describe is more than a decade old, and a lot has happened since then (we’ll write about the technology that we’ll be using later).

Dee-eN-A-what?

The science behind the LilBubome – Part 1.

In our previous blog post we spoke about the concept of the LilBubome and our idea to raise money for it through crowdfunding. Today we want to tell you a little about the science behind the project. We’ve mentioned that we want “sequence the DNA of Lil Bub”. Here, we explain what that means. Continue reading