Too much information – how to find something in BUBs genome

Hi everybody,

if you followed us here and here, you know that we have sequenced and mapped all of BUB’s genetic information.

Today we want to show you how we try to cut through all of the information that a genome holds to reveal the variants that we think made BUB BUB!

It’s all pretty exciting and also just a first glimpse on what we can find if we analyze even further! In any case. Have a nice weekend and fun watching – and as always: Let us know what you think or if you have any questions! We will answer. Pinky-promise (or in German: Großes Indianerehrenwort!)

 

Best

d

For the nerds of you: here’s a link to the software we used, it’s Platypus http://www.well.ox.ac.uk/platypus developed by Andy Rimmer and friends in Cambridge, UK.

For everyone – here’s the cool pic of the Hemingway Cats.

source (http://www.hemingwayhome.com/cats/)

 

A handful of BUB’s DNA

Some posts ago, we extracted some DNA from LilBUB to run a small test which revealed that BUB seems to be distantly related to Hemingway cats. However, to sequence her whole genome we require a bigger amount of DNA so we need to re-extract it from our blood sample (to remember the principles of DNA extraction check here).

But, how much DNA do you actually need to sequence the entire genome of an organism? Ideally 1 microgram should be enough. This is equivalent 0,000001 grams, which is the 50th part of a water drop. Pretty small amount, right? But in terms of our purpose more than enough!!!

After handling the sample to our sequencing facility, Norbert (one of the technicians working there) extracted BUB’s DNA. After the process he obtained a small volume of a water solution.

IMG_20150714_105915_HDR

Norbert in action

But how do we know how much DNA is contained there and its purity? This is determined by the ability of the DNA to absorb light, which in scientific jargon we call Absorbance.

To measure these parameters, a small drop of our water solution is loaded on a spectrophotometer. This machine emits ultraviolet light (wavelength of 260 nm) that pass through our drop, and is received by a photodetector. The less light reaching the photodetector, the more DNA is included in our sample. This analysis revealed that our sample contains up to 4 micrograms of DNA (well done Norbert!!!).

The spectrophotometer is also capable of measuring other contaminants that could be present in our sample, derived from a not so clean extraction. These contaminants absorb different types of light (with different wavelengths). The Absorbance ratios between different types of light revealed no contamination of proteins (260/280=1,82) and organic solvents (260/230=2,1), confirming the high purity of our sample.

150714_LilBUB-Gel

LilBUB´s DNA appears as a high molecular weight smeary band

Last, we checked the size of our DNA. For that purpose, a small part the sample is loaded in an agarose gel. When we apply voltage through the gel, our DNA will migrate from top to bottom. The bigger the size of our DNA, the slower it migrates. In our picture, you can appreciate that the size of our DNA is bigger that 10 kilobases, which means that it was minimally fragmented through the whole process. Taking into account that the genetic code is composed by bases (A, C, T or G) this means than, on average, we have long DNA stretches of more than 10000 bases!!!!

Next station, preparation of a DNA library for sequencing!!!!!

NerdTalk

OK people. We wanted to let you know how things sound and feel like in the lab. So this one is for those of you who are interested, have a little more background knowledge, or are scientists. These are some notes on what exactly we did to sequence LilBUB’s ZRS in a way that I’d write it in an email to Uschi or Dario or how I could reconstruct the experiment I did in my lab book. Continue reading

First bit of real research! How to make a LilBUBome – STEP 2. Sequence a target region

Hey All,

as we wrote you on Thursday, we’ve decided to do a lucky-guess experiment to look for mutations in a tiny part of LilBUB’s genome, a region called the ZRS. So, this week we’ve been working pretty hard to get a sample ready for sequencing, because May 1st is a bank holiday here in Germany. Well, it turns out I was too slow so now we’ll only get the result of what the exact DNA sequence of LilBUB’s 800bp ZRS is on Monday.

So, what did we do?

First, we extracted the DNA from some of the blood sample. We then used a technique called PCR to purify (and amplify) only the DNA contains the ZRS, so that we have a lot of material to analyze (here’s how it works).

After that, the first thing we checked is whether amplifying LilBUB’s DNA worked just like amplifying any other cat’s DNA (which I did as control in parallel – thanks to my neighbor Nadja for the sample). Turns out, BUB’s ZRS (remember, the part of the DNA that controls the number of digits) is the same size as that of the control. OK, nothing unusual so far.

The amplified ZRS of a control cat (left) and LilBUB (right)

But what about the actual DNA sequence? We sent only only the amplified bit of DNA in the picture above for sequencing (0.00023% of BUB’s genome) and until Monday we have to wait for the results. So let’s see, my guess is it’s like nothing unnormal in the sequence here as well.

Best

Daniel

The guessing game: searching the ZRS haystack for mutations

On Monday we had a state-of-the-crowdfunding meeting. We’re all really excited that we’ve already raised 28% of the money, and we’d love to share some of that excitement with you. So, after some discussion Daniel and Darío persuaded their boss to pay for a lucky-guess sequencing. This means we’re applying a magnifying glass at only a tiny part of the genome, because we’re hoping we might find something there. Basically, while searching for a needle in a whole bunch of haystacks, we’ve decided to focus on a single one (you can read more about different strategies for finding needles in genomic haystacks in our previous post: How to find a mutation? – Needles and haystacks).

The haystack we’ve decided to focus on is called the ZRS. The function and discovery of the ZRS warrants multiple blog posts in it’s own right, so let’s just say it’s a decent haystack to look at. First of all, it’s rather small: about 800 basepairs (building blocks of DNA), and only 0.000028% of the genome. Second, we know that there are a bunch of human patients, chicken, dogs and other cats with polydactyly, who all have mutations in the ZRS, so we figured: if we have to make a single guess this will be it.

poly

Polydactyly happens in many different species

Daniel started the experiments on Monday. We’ll keep you posted with progress!

– Uschi