Kickstarting a Eublepharus genome project.

[geckolabs]

For those of you who didn't listen to the late night leos show last night, or have been perusing around The Gecko Blog on facebook; We are finally getting serious about kickstarting a genome project.

Now before everyone goes crazy, let me clarify a few things:

1) In no way is this guaranteed to work, but at least it is finally going to be attempted.
2) There are a few end goals of this:
A) To be able to identify the locus (or loci) contributing to different traits (i.e.- exactly what loci is responsible for bell albino).
B) To be able to know the sequences of alleles identified as a result of (A) so that animals can be tested using simple polymerase chain reactions. (a.k.a.- NO MORE HIDDEN/UNKNOWN HETS)
C) Being able to test animals as described in (B) will also help to get back to starting and maintaining pure lines of each trait.

3) This isn't something that is going to be cheap, but will help keep our hobby thriving. Many small scale hobby breeders may be frightened by the idea of this. But to be honest, it is something that is going to take years of work to do, and offers nothing but positives for the hobby. As far as cost, it is still being developed and the associated equiptment/supplies being determined. But it is entirely feasible to be done for less than $150,000. Given the number of breeders there are, large and small, we should have no issue pulling together to fund this project.

4) This is not an attempt to scare anyone out of breeding by any means. It is an attempt to keep our hobby alive for generations to come, and pull traits back out and re-establish pure lines before things get too muddy to do so. Not only that, but this should be taken as an opportunity as the community to come together as one. There has been far, FAR to much bickering etc on the various FB groups, and we are only hurting ourselves in doing so. If we ban together to accomplish a project like this, we will be seen as more than "the reptile freaks," which is something the community desperately needs.

---------Now, what can you do to help?
In all honesty, unless you own animals that are derived from pure lines (and can prove it), then there isn't much you can physically contribute to the project, but you can still donate!

To the people who own pure animals, not combination morphs, but absolutely pure (i.e. ONLY bell albino, without any chance of carrying ANY hets), then contact me ([email protected]). I am going to attempt DNA extraction from fresh shed to see if sufficient enough of a sample can be derived from shed.

If shed is sufficient, then that is great news, as all I would need is multiple samples of fresh shed from a pure animal of each trait/subspecies.

If shed is not sufficient, then things get messier. But we will cross that bridge when we get there.
---------------------------------------------------

Let me know if you have concerns, comments, questions, etc., as I am sure there will be plenty. I can best be reached either through FB (Dillon Damuth- add me, I don't bite), or email ([email protected]).

Ps, Maybe admins can make this thread a sticky???

Thank you all!

 

[Music City Geckos]

If you want pure lines, John at Gecko Boa comes to mind.

 

[lillith]

+1

BTW, I'm a very small scale breeder, and I'm not afraid of this at all. It would be a huge relief, actually.

As far as I know, my bells and my tangerines are non-het. I'd be happy to send shed over. Where do we send it? Do you need a whole shed, or a partial (if so, what is the minimum % partial that would work, based on adult size? i.e., how many square inches per gecko needed?) would sending them over individually sealed in new ziplock bags work?

 

[geckolabs]

As a simplistic explanation for those who aren't too genetic savvy:

Ok, so to simplify for explanation's sake, lets say the genome is a linear, piece of DNA. If we sequence and put together this genome, so that we can segregate and be able to know one loci from another (see panel A), we will be able to compare it to animals with traits that deviate from the 'wild type.' Therefore, if we take, for example, a Bell albino, and look at its genome, we can compare the various loci (panel B) to determine which location has an allele variant (aka, the bell allele.) Then we can continue to do this for other traits, such as murphy's patternless (panel C) and so on. Once we have identified all of the various alleles, we can then have these alleles sequenced. Once we know the sequences of the alleles contributing to each trait, primers can be designed so that they can be used in PCR reactions to amplify specific sequences. Therefore, given an unknown animal (panel D), where for example and animal is sold as an albino patty, it can be tested for exactly which strain of albino. Since we now know the sequence of the albino alleles and have primers designed (panel E), we can use the primers to add to DNA and run a PCR reaction. When we run these reactions on a gel, the banding pattern will inform us exactly what alleles the animal in question carries. So if you look at the gel, we can see the first lane is just a reference ladder, to tell the size of the DNA fragments that were amplified in the reaction, but the lane 2 is a sample from a known bell, lane 3 from a known non-albino, lane 4 a known het bell, and lane 5 the unknown 'albino patty.' Looking at the banding patterns, the known bell has only one band, meaning it only has one allele variant (even though there are two copies. the two copies would run at the same size, leaving only one band.) The known non-albino has only one band as well, but that band is for the wild-type allele at locus 'A' (refer back to panel A.) Then a known het bell will give two bands, one for the albino allele, and one for the normal allele. The animal in question gives only one band, meaning it is either homozygous for bell, or homozygous for normal, but comparing band size, we know that it matches the band size for the bell allele, allowing us to conclude the animal is a bell patty.



This could eventually be done for any animal, and for all of the traits. Meaning no more unknown hets on the market, no more possible hets, and so on.

I'm not sure how well you can read the figures, so if you have trouble, email me and I can send the full resolution figures.

 

[geckolabs]

As far as I know, my bells and my tangerines are non-het. I'd be happy to send shed over. Where do we send it? Do you need a whole shed, or a partial (if so, what is the minimum % partial that would work, based on adult size? i.e., how many square inches per gecko needed?) would sending them over individually sealed in new ziplock bags work?

That's good to hear. I think some smaller breeders may get frightened at the idea of the hobby becoming more 'industrialized.' But to address anyone's possible fears further, this in no way is meant to industrialize the hobby. It will still be each breeder's choice whether or not to test their animals. If an individual breeder chooses not to, for a small additional fee, it would be reasonable for the buyer to be able to request a sample be sent to test for assurance and piece of mind.

Keep in mind, that once the allele variants are known, the testing will be quite cheap, and large scale breeders who have the money to invest may purchase the associated equipment and offer the services to customers and others, alike.

The extent to which this can benefit our hobby and clean up the genetics is endless!


To address your question about where/how to send them, shoot me an email @ [email protected]. Ziplock bags should be fine. And a few good sized pieces of shed per animal should be more than enough. I will have a better idea of how much will be needed once I know whether or not shed will be sufficient enough of a sample. When I know that, I will post an update and disseminate the information regarding the hows and wheres of shipping

 

[indyana]

Not frightened of the idea of DNA testing unless the cost is astronomical. I'll keep an eye out when you start collecting for the fund. Good luck, and thank you for seriously considering this project.

 

[geckolabs]

Not frightened of the idea of DNA testing unless the cost is astronomical. I'll keep an eye out when you start collecting for the fund. Good luck, and thank you for seriously considering this project.

Thank you for the support, Rachel!

And to address costs, here are a few good points that I should mention:

What i envision as a result of this project, is a handful of breeders that invest in this startup equipment (thermocyclers, primers, PCR reagents, DNA extraction kits, Gel electrophoresis chambers, etc.)

Cumulatively, to make that investment, it should cost no more than $2000. I know that is a hefty number, but bear in mind, the thermocycler and the extraction kit will be the most expensive components. The thermocycler will last a great deal of time, given it is used correctly, and the kit will be suitable for 250 reactions. The PCR reagents would be somewhat costly, but not as much as the previous two components. Now lets say a potential buyer wants to have an animal tested- be it from the breeder who owns the equipment, or another- I see a developing hobby where said buyer could pay an additional fee to have the animal tested to confirm or eliminate possible hets.
*Keep in mind that each breeder that invests in the equipment will be entirely able to declare their own rates for the service*

Ideally, if the breeders offering the tests charge just $20-30 for the test, you could have the animal tested, and it doesn't take long at all. it is something that can be done in a matter of hours within receiving the sample. The proposed rate would allow breeders to cover their investment within the time of going through one 250 reaction kit, and have the funds to purchase another kit.

 

[VampyreByte]

When this gets up off the ground my wife and I will be all for puchasing the equipment to do our own testing! I'm looking forward to this. I love the science and genetics of this hobby! I'm no scientist or geneticist, just a humble power plant electrician with a love for science topics and reptiles!

Damn this is going to be fun! I can't wait!

 

[geckolabs]

When this gets up off the ground my wife and I will be all for puchasing the equipment to do our own testing! I'm looking forward to this. I love the science and genetics of this hobby! I'm no scientist or geneticist, just a humble power plant electrician with a love for science topics and reptiles!

Damn this is going to be fun! I can't wait!

That is exactly the enthusiasm I like to see!

Not everyone in the hobby, hell, very very few in the hobby are scientists. Thats part of why I want to bring something new to the table to help develop the hobby. I know this idea has been bounced around before, but not enough people were showing the enthusiasm we're seeing this time around, and noone wanted to do the benchwork.

Now we have the enthusiasm and the manpower. There's no excuse to not pursue it this time!

 

[VampyreByte]

Exactly, and i'm not sure we had someone around with a background in actual genetic sciences or lab work with it. Or just no one that was willing to take the initiative like you are!

I'm sure a lot of people might be worried what genes could be present in their animals. I find it exciting though, even if my animals had discrepancies. We might never have possible hets again after this takes off!

In my opinion this might scare the bigger breeders more so than us smaller ones, just because they have more to lose if they find discrepancies in their animals genetics.

 

[geckolabs]

This is certainly becoming very real, very fast. I am currently talking to a technician for the company through which my lab uses for sequencing to work out kinks in the design and get a price quote.

 

[rothsauce]

I'm very much liking the sound of this.

It would end the quips regarding purity, and help breeders unlock new combinations in the process. Plus authenticity could certainly be proven and recorded, fewer doubts and shady deals when it comes to higher-end lines.

As a non-breeder, this wouldn't impact me... at all really, but the prospect to see something really come of this is very exciting!

 

[HerpMorphs]

Awesome stuff Dillon. Can't wait to see more

 

[geckolabs]

The general consensus is that people are most worried about the 3 strains of albinism. So here is an idea that is in the works:

Getting the initial sequencing done, and identifying the loci associated with the 3 traits, and then start offering testing, at slightly higher prices, and use all of the proceeds to fund the remainder of the project.

I am hoping to get initial fundraising kicked off in the spring, since it is the end of the season and everyone is currently broke.

Comments? Concerns? Questions?

 

[Warren_Booth]

The simplicity of this scares me a little as it is clear that this project is far from developed and likely completely misunderstood. I do this for a living and have been and continue to be involved with genome projects and gene identification. Let me outline my concerns here.

1) A genome is not available for Eublepharis macularius and I am not aware of one even in the pipeline. We have the 10k project, and it has been listed, but that is years away from even being started. Will it be??? Possibly, as like any gecko we may learn more about tissue regeneration from understanding the genes and gene interactions involved. But, that is a long way off. A genome will cost around $150,000. What will that get you. Is should get you a draft genome, but not an annotated genome. That is the hard part. Having a line of sequence is nowhere near understanding what genes are functioning and which not. The reason being is that DNA is not all coding. We have introns and exons present in DNA sequence.
2) Its not just a case sequencing the genome and instantly knowing what the gene is for bell albino. To even narrow down that region will require another $15,000 to $100,000 work (for a single gene). This would require something like RADseq to identify the regions possibly linked to a trait. From there, you then need to identify the gene (splicing the intron), and would then need to undergo gene-expression studies to see if any of these genes actually are related to the function of interest. That, is for a single gene. At that point in time, if you happen to narrow it down to a single gene, then you can design primers. What is the likelihood of getting to that point???? Very slim. A lab that would get involved in this would need to pay multiple researchers. So your are looking at anything from $35,000 to $105,000 per person per year. Lets look at the team that was involved in the termite genome, form which we now have a draft and have "narrowed down" some genes that might be involved in caste determination. I think it was 44 researchers. Just for a small subset of genes. That took 4 years of 100% dedication of research to a project.

3) To set up a lab to test the samples will not take a cumulative $2000. Let me give you an idea of what I spent to equip my lab 2 years ago. 4 thermalcyclers - $20,000. 2 x 24 tube centrifuge - $1450 each, plate spinner - $6400, two gel rigs - $650 each. Additional gel casting combs - $240, powerpack to run gel rigs - $450. Pipettes - ~$350 each, 3 sets bought, 6 per set. From there, you then have extraction kits - generally around $500 per 250 samples, agarose, several hundred dollars per 100grams, sizing ladder - about $100 per 100 lanes. To visualize your gel you will need an imaging system. I paid $9500 for mine. So, that takes you from tissue to DNA on a gel. How about a genome. Well, you can buy a small genome sequencer for RNAseq - We paid $150,000 for our Illumina MiSeq. You would need much more power for a genome. So, say you get to use an Illumina HiSeq. You are talking a lab that has laid out $500,000 for it. Genome DNA library construction, sequencing kit, etc. Costs around $3500 to $5000 per run. For sufficient coverage you are looking around $80,000 to $100,000 of sequencing. Then you have genome assembly. Programs that will get you started, e.g. CLC genomic workbench will run you $5,000 for a static license with one year of service support. Hmmm, from there, there is the joy of genome annotation. Many many years of work depending on what you want to do. But, with the genome you could then do RADseq to try and narrow down your regions of investigation. As mentioned - $15,000 to $100,000 depending, per gene.

It is sadly a pie in the sky dream to think that you can simply extract DNA from a gecko, sequence its genome, identify a gene, PCR it and tell if your animal is a het. Sorry, but you need to do A LOT more research on this actual topic to understand the process, the costs involved, and the limitations. Oh, and the likelihood of identifying a gene. You know that the human genome project has cost $100 billion, has taken over 10 years, and still has not identified all of the genes at play. If you find a lab that will do this for free, so that they can make $15 or $20 a gecko to test what gene it is... tell me who they are and I will form a collaboration with them. They can do my work for free and I will then send a few samples to them to test, lol.

Sorry, but this is not even close to being a viable project.

Regards,
Dr. Warren Booth
Booth-lab

 

[geckolabs]

And people like you, who are pessimistic about the project, are the reason it has been dismissed in the past.

I have already been in contact with representatives and technicians. The end goal is to identify varying regions specific to each trait, not to specifically narrow down exactly what the gene is, or what it does. At this point, the collective community does not care about the molecular function of how gene X contributes to phenotype Y. This is exclusively to be able to identify regions with variability between pure examples of each trait through subtractive comparison of the resulting data.

I am well aware of the processes involved, and would not need to worry about lab startup costs, as I already work in a lab that has the equipment.

At this point, yes, the project is far from fully developed and still in infancy, hence the title of the thread if you cared to read it.

In conclusion, you can take your pessimism elsewhere as we push the project forward.

 

[Warren_Booth]

Hi Dillon,
To respond to your very negative response, I, in no way was being pessimistic in regards to your idea. I think its great to have a dream. However, in this genomic era you need to be versed in how exactly this goal can be met, and I do not feel that you are.

I want people to realize that it is certainly not as simple as you make out and that the cost is much much greater than your $2000 estimated outlay. I have already been in communication with a ball python breeder to map out regions for an unusual trait. The cost we came to was $100,000, to identify a single trait.

To identify variants requires you to understand the genomic region under control for these traits, otherwise you will not be able to create markers to test this. Variation can be generated anywhere in a sequence through single nucleotide polymorphisms. These may have no impact on the actual trait you are interested in.

With an assembled genome, you can then use RADseq to determine regions of variability within and among your specific traits. But, to get to that point you are still talking around $200,000. The reason we can do the ball python one for $100,000 is because we have a genome that we can use as a scaffold for assembly. There is nothing comparable for Eublepharius.

I know this might sound pessimistic, but this, financially, is much beyond any kickstarter program, and indeed most DNA screening labs out there. Whether you like this or not, this is gene discovery, then the generation of probes after that. This is what I do for a living and am pretty versed in the ins and outs.

Warren

 

[VampyreByte]

HURRY UP DILLON!

LETS GET THIS GOING!