Jump to content

e-DNA Sampling For Sasquatch


Recommended Posts

Madison5716
On 6/23/2020 at 3:30 PM, Catmandoo said:

Jonah Ventures has  'diet test kits ' that  are used for herbivores and carnivores. Worth a look.  Send in a scat sample!   Turdology is looking up. They take PayPal.

 

Very interesting! Another item for the tool box potentially.

Link to post
Share on other sites
BobbyO
SSR Team
On 6/22/2020 at 4:49 PM, hiflier said:

Me collate the reports from the last five years? I have enough on my plate just with this. If you don't wish to corral the reports yourself and bring the data here, then maybe someone else is willing? 

 

 

Ask and you shall receive ! ;)

Link to post
Share on other sites
hiflier

You, sir, would bring a tear to my eye :) 

 

I probably should take this opportunity to present a couple of updates. These are responses that I have received regarding my NOTCH2NL concept:

 

1) "It would be a great study, but I am afraid you will need a good fresh sample of blood or tissue or bone to extract enough nDNA to sequence these genes.  I read about the genes and had no other thoughts.  Best of Luck.  If you get an adequate sample you should try a whole genome sequencing to answer many more questions."

 

Followed up response:

 

"Your genes are nuclear genes, but there is a 1000+X advantage in mtDNA, so much better chance to capture in an e DNA experiment.  It doesn't hurt to look, however.  I do think that a well designed eDNA project involving collection of water samples could reveal an unknown primate."

 

And this is a response from a different source. A lab in fact:

 

".....there are two technical issue that impacts your suggested approach.
1)
Species ID approaches typically use mitochondrial DNA, as there can be dozens to hundreds of mitochondria per cell. Importantly, this means that mtDNA is at least one (but probably more) order of magnitude more common in the environmental than nuclear DNA.

Given rare species, by definition, are at low density, their DNA in any given environmental sample is present in low concentration. For quantitative PCR, the limit of detection is around 1x10^-5 or 1x10^-6 nanograms per microliter. The concentration of mtDNA for rare species is typically at or below limit of detection, so we have to sample in ways to get the DNA concentration above the detection threshold. High volume filtered, technical replicates etc.
We are investigating ways to reliably use nuclear DNA from environmental samples, but this isn't something we recommend at this time.

2)
Environmental samples are "pools" of all the DNA present. Many different organisms and/or multiple individuals of the same organism. So you either have to target the species of interest using qPCR or you have to conduct a metabarcoding analysis, which separately analyzes each DNA strand present. As there is no qPCR assay specific to Bigfoot, metabarcoding is the only choice for water samples. [Soil is a different story] Currently, given the sequencing technology, I think you'd have to conduct meta
(data), but use the NOTCH2NL marker for amplification to pull the locus of interest from the "pool" of DNA.

We can process disc filters, although we recommend sterivex filters for rare DNA at low concentration."

 

So, as you all can see, I have been trying to do my part as far as laying the groundwork, not only to find support for the idea, but to also find a facility that would be open to testing samples. Something that I'm continuing in doing because one lab may not be sufficient if any samples succeed in getting a positive result. So far there seems to be some good support for the NOTCH2NL approach. It has taken months to nail down positive responses like these. Nearly all inquiries have otherwise gone ignored.

 

 

  • Thanks 1
  • Upvote 2
Link to post
Share on other sites
norseman

Exciting!!!  
 

What I’m reading is that if they know what they are looking for (unknown primate) they can adjust their studies to accommodate the search?

 

So.....there IS a way forward concerning this research!

 

By high volume? How much water are they talking about I wonder?

Link to post
Share on other sites
gigantor

Great job Hiflier!

 

30 minutes ago, norseman said:

How much water are they talking about I wonder?


If you want detection you need:

 

3 hours ago, hiflier said:

For quantitative PCR, the limit of detection is around 1x10^-5 or 1x10^-6 nanograms per microliter.

 

Nanogram =  billionth of a gram

 

Microliter = Millionth of a liter

 

1000000 x 0.000000001 =  0.001 grams per millionth of a liter. 

 

That's a lot concentration me thinks. A millionth of a liter is pretty small I suppose. We would have to filter a lot of water....

 

We could also do this in soil if we find a nest.

 

Link to post
Share on other sites
norseman
12 minutes ago, gigantor said:

Great job Hiflier!

 


If you want detection you need:

 

 

Nanogram =  billionth of a gram

 

Microliter = Millionth of a liter

 

1000000 x 0.000000001 =  0.001 grams per millionth of a liter. 

 

That's a lot concentration me thinks. A millionth of a liter is pretty small I suppose. We would have to filter a lot of water....

 

We could also do this in soil if we find a nest.

 


I would be curious to know when they discover a new species or even detect extant ones? How many liters of water does it take on average?


Are we talking 10/100/1000/10x100?

 

Link to post
Share on other sites
gigantor
23 minutes ago, norseman said:


I would be curious to know when they discover a new species or even detect extant ones? How many liters of water does it take on average?


Are we talking 10/100/1000/10x100?

 

 

 

It depends on how much dna of the subject there is in the water. If you get water from a running River, there's probably very little of it and you would need to filter a lot of water, hundreds of gallons or more.

 

If you get water from a puddle where Bigfoot just took a leak, then a jar of water would be enough. 

 

Something like that.

Link to post
Share on other sites
hiflier
Posted (edited)
11 hours ago, gigantor said:

Great job Hiflier!

 


If you want detection you need:

 

 

Nanogram =  billionth of a gram

 

Microliter = Millionth of a liter

 

1000000 x 0.000000001 =  0.001 grams per millionth of a liter. 

 

That's a lot concentration me thinks. A millionth of a liter is pretty small I suppose. We would have to filter a lot of water....

 

We could also do this in soil if we find a nest.

 

 

Math? Wouldn't that mean 0.001gms per millionth of a liter = 1 gm per 1 thousandth of a liter, or 1cc? That's would be a lot of material per cc of water. Or did I go the wrong way? Also, the figures he gave are the absolute bottom limit for detecting anything.

 

 

Edited by hiflier
Link to post
Share on other sites
gigantor
1 hour ago, hiflier said:

 

Math? Wouldn't that mean 0.001gms per millionth of a liter = 1 gm per 1 thousandth of a liter, or 1cc? That's would be a lot of material per cc of water. Or did I go the wrong way? Also, the figures he gave are the absolute bottom limit for detecting anything.

 

 

 

I think that is correct, but I hope  I made a.mistake somewhere.

 

1 x 10^6 nanograms. =  1,000,000. nanograms.

 

1 nanogram = 1 billionth of a gram = 0.000,000,001 grams

 

1,000,000 x 0.000,000,001 = 0.001 grams.

 

This can't be right. That is a lot of material, it almost defeats the purpose.

 

 

 

 

 

 

Link to post
Share on other sites
hiflier
Posted (edited)
15 hours ago, hiflier said:

1x10^-5 or 1x10^-6 nanograms per microliter

 

Methinks the minus powers might be the issue? I get lost when on the right side of the zero.

Edited by hiflier
Link to post
Share on other sites
gigantor

Oh, yeah. I didnt see the negative sign!

 

Thats a huge difference.

 

1 x 10^-6 = 0.000,001

 

0.000,001 x 0.000,000,001 =   0.000,000,000,000,001 grams

Link to post
Share on other sites
norseman
12 hours ago, gigantor said:

 

 

It depends on how much dna of the subject there is in the water. If you get water from a running River, there's probably very little of it and you would need to filter a lot of water, hundreds of gallons or more.

 

If you get water from a puddle where Bigfoot just took a leak, then a jar of water would be enough. 

 

Something like that.


Hmmmm.... 

 

100’s of gallons isn't doable for sending a sample UPS to a lab.

 

And if Im watching Bigfoot take a leak in a puddle? Im getting more than a water sample...

 

Are they setting up a lab on the side of a river somewhere in tents? Running a pump and a hose inside?

  • Haha 1
Link to post
Share on other sites
gigantor
4 minutes ago, gigantor said:

Oh, yeah. I didnt see the negative sign!

 

Thats a huge difference.

 

1 x 10^-6 = 0.000,001

 

0.000,001 x 0.000,000,001 =   0.000,000,000,000,001 grams

 

 

0.000,000,000,000,001 grams

 

I'm headed to the beach or my wife will kill me...

  • Haha 1
Link to post
Share on other sites
hiflier
10 minutes ago, norseman said:

And if Im watching Bigfoot take a leak in a puddle? Im getting more than a water sample...

 

Ya Baby!

 

10 minutes ago, norseman said:


Hmmmm.... 

 

100’s of gallons isn't doable for sending a sample UPS to a lab.....Are they setting up a lab on the side of a river somewhere in tents? Running a pump and a hose inside?

 

Good logistics question. I have already asked the lab what they would do in our situation as far as procedures for sampling. And they already know what we're after so if I can maintain a dialogue then maybe we'll have our answers. And you know? All I've ever wanted from anyone are answers to questions. I mean, good grief, it's not like I want the Moon here. But we need specifics or else things could end up being a horrible and expensive trial and error. So how would a lab person conduct the project in the field if they were me? That's the question I left them with. It's 4th of July weekend and the lab may have the day off so I don't expect any dialogue until next week earliest. And you know me, Norseman, I'll stay with it until we have what we need to know.

 

They also said in their responses above that "soil is a different story". We need to know that "story" as well. Hey, no one said this citizen science stuff was going to be easy, although I think it would be for normal animals. That said, what they said was that metabarcoding for ALL animals in the sample would be a good way to go as far as looking for NOTCH2NL markers. We could use some clarification on that aspect of things.

  • Thanks 2
  • Upvote 1
Link to post
Share on other sites
BlackRockBigfoot

@hiflier

 

Thank you for all of your perseverance and hard work!   

 

If soil is the route that we need to take...well, @Madison5716, @NorthWind, myself, and a few others may will still be able to collect samples.  I would assume that a fairly fresh print from a bare foot would contain discarded skin cells and would be prime material for testing.  You can never tell what the future holds, but I currently have two areas where we have found prints recently.  Once we determine the proper collection techniques and tools I can collect soil if the opportunity presents itself again.

Link to post
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
×
×
  • Create New...