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e-DNA Sampling For Sasquatch


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hiflier
28 minutes ago, norseman said:

I personally would like to see samples go through a transparent chain of custody WITH results posted in the end.

 

 

I agree and would like to see transparency as well. But the most important transparency would be, as you say, the results from any testing. The SSR database could be sorted for the last five years per NatFoot's suggestion? But I also think leaning toward the reports from our own researchers would have high value. No one has to give specifics on location but information on what has been observed would be helpful. You mentioned Sullivan Lake. If activity is relatively current to the extent that it would seem creatures are in the area then, yes, it would be a candidate for sampling- especially close to the mouth of a stream where it feeds into the lake. Something where water might not be moving fast enough to carry a lot of heavy sediments? I think the perfect set up is a small waterfall type situation where one could collect the smooth water coming over a rock while standing downstream from  it.

 

Here is the type of filter I would use to virtually eliminate Human contamination. One doesn't need to handle and fold a filter membrane and try to get it into a test tube without touching it. This product allows sampling and then the whole self-contained unit gets sent in. The company is Smith-Root out of Vancouver, WA.: https://store.smith-root.com/edna-045-um-filter-pack.html

 

We could also use some ideas on how to collect sample just using some kind of gravity feed set up. Mainly because the electric vacuum pumps, either hand held, portable drill operated, or manually operated are expensive. This is why I have mentioned universities that do citizen science that will train, provide sampling equipment, test and show their results, all for nothing. But in lieu of that, a cheaper way to force water through Smith-Roots filter cartridge might be good to hash over.

 

Something that might take two people like the filter pack attached to a sterilized tube mounted to the bottom edge of a five gallon white bucket. The bucket can be sterilized with a bleach solution (the outside doesn't need to be) and capped until on site. Rinse the bucket a bunch of times under the waterfall  to clear the bleach and then one person sticks the bucket under the waterfall, and the second person holds the tubing and filter. Stand down stream and wear gloves to keep Human DNA out of the mix. The hard part will be waiting for the water to completely empty (gravity?) through the filter. A couple of quarts is all all that would be needed so it's not like someone has to stand there and hold five gallons of water (40 lbs.!) until it all drains through. The more water, though) the more gravity can help to force the water through. Beyond that I think the cheapest (still not cheap) might be a cordless drill with a peristaltic vacuum pump attached. 

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hiflier
2 minutes ago, gigantor said:

Lets figure out the details and procedures first. We'll figure out the money part later, I'll help with that.

 

Thank you, gigantor, always the wise one :) Certainly there's groundwork to be done behind the scenes. And then there's making sure good field techniques are learned. I could use some practice there myself to make sure I keep myself out of the equation when taking samples. Because, YES!, I will be taking samples too if any recent reports say "go". And then there's lining up the right materials to use in the field. I think most of us have been in this for the long haul so taking the time to get things right won't be an issue. We have from here on out, in any season, to pull this off. Dang, I'm almost ready to sell my thermal imager.......um......well.....maybe not quite yet ;) 

 

One very serious thought that has been creeping into my mind in in regards to a dead Sasquatch out there somewhere who's DNA has been leaching into the soil and surrounding watershed. If that's the case then e-DNA will help greatly in us not having to look for the needle in the haystack.

 

P.S. Looks like the BFF is moving further into the realm of hard science.

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hiflier

While some research on my end is being done, how about let's discuss the mechanical side of data collection. The USGS .pdf I uploaded has a lot of good info on collection materials and tools but then mush of what is listed is quite expensive. No surprise there since the USGS has deep pockets, but I have questions about other types of equipment that would put me in the poor house. One thing that should get discussed, in lieu of a gravity feed-water set up is vacuum pumps. Out of all of any sample system's components that is the relatively big ticket item.

 

A vacuum pump, manually operated or not, does not have to be sterile since it is attached AFTER the filter. So it's not like one is trying to push water through the filter as much as pull the water back through it. Of course, such a device is only needed when sampling water. Soil is another animal altogether. Needless to say, there's a lot more info on water sampling than soil sampling. In a nutshell, soil sampling has a lot more going against it where natural factors affecting the quality of DNA in soil come into play. More on that later.

 

So, vacuum pumps: Here's one as part of a portable drill set up: https://genidaqs.com/wp-content/uploads/2018/04/Genidaqs-eDNA-sampling-procedure-201708.2.pdf  I'm posting the article mainly to reinforce the technique of proper sample collecting. NOT to promote that Easy-Load drill pump head. That little baby goes for over US $400!  It seems as if once e-DNA get attached to a product the price quadruples. We can do better. A handheld pump like a car mechanic uses goes for between $16 - $35. I do know that there is a certain vacuum pressure that is more favorable for filtering, and that consistent pressure is also more desirable.

 

A handheld, even a cheap one, has to produce the right amount of water pull without back washing into the filter and ruining the sample. So a vacuum needs to make sure that water flows under a fairly consistent negative pull from whatever pump gets used. Pretty sure stopping and starting the flow will contaminate the sample. A mechanics hand held pump may not be the thing to use for as sensitive an operation as looking for Sasquatch DNA. Personally, I do not want to be too cheap for the discovery of a last century and a half. And, one usually gets what one pays for. Will a lab want to know what my collection methods were? Maybe. But the important thing is...I will know, and I need the confidence I did it right.

 

Hey, this is a smart crowd and some people know things that I don't so some input on this particular mechanical feature would be welcome. Both for reducing cost as well as having the desired function  dynamics for water sample collection.

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NatFoot
4 hours ago, norseman said:

We are going to have to allow Hiflier the time to find a lab willing to do E DNA samples. And enough time to raise the money to pay for the lab. And the chosen lab is going to have to line researchers out on protocol for taking the samples.

 

I think the Go fund me idea is an awesome idea.

 

I personally would like to see samples go through a transparent chain of custody WITH results posted in the end.

 

For example.

 

/////////////////////////////////////


Sample # 2367

Researcher: Bob Smith

Contact info: Address/Phone number

Date: 10-25-20

Location: Pend Oreille River, Lat/Long

Comments: I have recorded audio of whoops in this location before. SSR has reported 3 sightings within a 10 mile radius over the past 5 years.

 

Sample # 2367

Collector: Bill Jones

Contact info: Address/Phone number 

Date: 11-2-20

Lab: Horizon Laboratories Inc. 12345 Pine Ave, Denver, Co 76543

Comments: Sent off Fed Ex ground today.

 

Sample # 2367

Researcher: Bob Smith

Date: 12-16-20

Lab: Horizon Laboratories Inc. 12345 Pine Ave, Denver, Co 76543

Lab Results: Rainbow Trout, Northern Pike, Beaver, Black Bear, Whitetail Deer.

Comments: Did not sequence plant life per request. 
 

///////////////////////////////////////////

 

I think many researchers get disillusioned over the years by sending in samples only to not hear anything back at all. I think in order to keep people engaged people need feedback. Even if it’s not the target species we are looking for. People will feel better about their work if they see results. If this technology is as good as they say it is? Using the grass roots approach? Hundreds of researchers submitting hundreds of samples? Is going to cover a large chunk of real estate fairly quickly. If the beast exists? It cannot hide forever. And anyone can put on some rubber gloves and collect a water sample. It’s not rocket science.

 

We have been stagnate for awhile now. Obviously what I’m doing isn’t working. This would be a new powerful tool for researchers to put in their tool belt. Who knows? We may get lucky!

 

 

 

I agree. If it got that serious, I'd imagine we would need to move that to the private/premium part of the forum.

 

Really like your additional suggestions though.

46 minutes ago, hiflier said:

While some research on my end is being done, how about let's discuss the mechanical side of data collection. The USGS .pdf I uploaded has a lot of good info on collection materials and tools but then mush of what is listed is quite expensive. No surprise there since the USGS has deep pockets, but I have questions about other types of equipment that would put me in the poor house. One thing that should get discussed, in lieu of a gravity feed-water set up is vacuum pumps. Out of all of any sample system's components that is the relatively big ticket item.

 

A vacuum pump, manually operated or not, does not have to be sterile since it is attached AFTER the filter. So it's not like one is trying to push water through the filter as much as pull the water back through it. Of course, such a device is only needed when sampling water. Soil is another animal altogether. Needless to say, there's a lot more info on water sampling than soil sampling. In a nutshell, soil sampling has a lot more going against it where natural factors affecting the quality of DNA in soil come into play. More on that later.

 

So, vacuum pumps: Here's one as part of a portable drill set up: https://genidaqs.com/wp-content/uploads/2018/04/Genidaqs-eDNA-sampling-procedure-201708.2.pdf  I'm posting the article mainly to reinforce the technique of proper sample collecting. NOT to promote that Easy-Load drill pump head. That little baby goes for over US $400!  It seems as if once e-DNA get attached to a product the price quadruples. We can do better. A handheld pump like a car mechanic uses goes for between $16 - $35. I do know that there is a certain vacuum pressure that is more favorable for filtering, and that consistent pressure is also more desirable.

 

A handheld, even a cheap one, has to produce the right amount of water pull without back washing into the filter and ruining the sample. So a vacuum needs to make sure that water flows under a fairly consistent negative pull from whatever pump gets used. Pretty sure stopping and starting the flow will contaminate the sample. A mechanics hand held pump may not be the thing to use for as sensitive an operation as looking for Sasquatch DNA. Personally, I do not want to be too cheap for the discovery of a last century and a half. And, one usually gets what one pays for. Will a lab want to know what my collection methods were? Maybe. But the important thing is...I will know, and I need the confidence I did it right.

 

Hey, this is a smart crowd and some people know things that I don't so some input on this particular mechanical feature would be welcome. Both for reducing cost as well as having the desired function  dynamics for water sample collection.

 

If this requires large purchases, or even for one to get a pump to lug into the remote woods - this already seems less realistic. Without writing a paragraph - why doesn't a simple set of water samples in hand collected vials not work?

 

If the DNA is in the water, it's in the water - right? Human DNA will be in the source most likely as well so why can't my hand just dip into the creek/pond and fill a vial with water?

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hiflier
6 minutes ago, NatFoot said:

If this requires large purchases, or even for one to get a pump to lug into the remote woods - this already seems less realistic. Without writing a paragraph - why doesn't a simple set of water samples in hand collected vials not work?

 

If the DNA is in the water, it's in the water - right? Human DNA will be in the source most likely as well so why can't my hand just dip into the creek/pond and fill a vial with water?

 

Bottom line? more bang for the buck. And I have to say that if what you say would work then it would be the method science would use. The idea of filtration is to gat as much DNA onto the filter as possible which, as you know, will cost the lab when they go to do the filtering themselves. Which they will have to do in order to extract the DNA from the water sample. Filtering a liter or more of water in the field will yield much more product than what is in a test tube. That's my answer, maybe someone has a counter and if so I am all ears.

 

On the subject of filtering, I have mentioned the single use filter packs from Smith-Root. The reason is clear when you read this 2019 article from the UK:

https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/2041-210X.13212 The self contained, self desiccating filters are the only way to go for reducing contamination and being able to preserve a sample inside the filter pack for up to six months at room temperature. So I could cap the ends and toss the thing into a drawer and, hopefully, not have a senior moment and forget that it's there. But for $15 against the hassle of packing in tubes of ethanol on-site to transfer the filter material into for preservation? That's too much for me to want to manage and feel like I wouldn't make a mistake along the way. These filter packs are a no-brainer for that reason alone.

 

It would mean $100 to take six samples, plus the cost of the other equipment, but there is a way around that. If several researchers live reasonably close by then sharing the mechanicals would help offset the initial outlay. Filter packs then could be purchased as needed. I also think our credibility on the lab-end of things will be enhanced when these packs are received. I can't help but think of the skeptical mindset of a technician WHO MAY THINK THEY ARE BEING HOAXED WHEN TESTING A LOOSE SAMPLE IN A TUBE! We have no room for errors or losing credibility before we can even get the sample to the GenBank.

 

Speaking of GenBank, we should probably get into that aspect of the equation sooner than later in order to assess where our possibilities for discovery may lie, and how to address the conundrum of not having Sasquatch DNA data to match samples to.

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NorthWind

Lots of peristaltic pumps available on ebay. Some are less than $20. https://www.ebay.com/sch/i.html?_from=R40&_trksid=m570.l1313&_nkw=PERISTALTIC+PUMP&_sacat=0

 

Now what about soil? For example, finding prints like Madison and I do. No water in them, but still probably loaded with eDNA if they are what we believe they are. Can that soil just be collected in a little zip lock with a clean and sanitized tool?

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Twist

What is the plan for E-DNA results? They come back with human DNA.   I’m assuming this vs. contaminated, Im assuming you get a result list back of known DNA.   All the sample results can come back human, do you even know for sure if this Upper/lower gene is identifiable with what we have coming back in regards to results? Do we know who/how they can check this?

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BlackRockBigfoot

From the little bit of research that I have done so far, a common vacuum hand pump would suffice for our purposes.  They can be found for under 20 bucks.

 

Say, $20 for a vacuum hand pump, $60-$90 dollars for the filter packs that Hiflier recommends.  Gloves, storage bags, bleach and other various stuff for decon...

 

It's doable.

 

I think that we need to agree on established best practices and standardized equipment so that we are maintaining the integrity of the samples across the board.  I am going through the links that Hiflier has provided.  e-DNA is not something that I have given more than a passing thought, so I am allowing his expertise to be my guide.  

 

@hiflier any recommendations on soil sample collections?

 

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hiflier

@NatFoot You're welcome.

 

@NorthWind Of course soil can be taken. It's what was collected at the OP nesting site and is also what is collected in caves like in Siberia ;) The methodology is perhaps something you could research and bring here? I was going to go into it a bit later myself anyway, so up to you.

@BlackRockBigfoot Well this soil thing seems to be BIG, LOL. I wish Dr. Meldrum and Dr. Disotell would come here and walk us through all of this. I really do. Okay, next up, soil sampling. And thank you for the shopping list. I should order some of that stuff, get out in the field, and do some practicing.

 

@Twist Okay, great question, and I hope I can answer it. This brings us to the GenBank issue. There's one thing that Dr. Meldrum and Dr. Disotell each said in their separate podcast interviews about the test results from the nesting site's E-DNA soil sampling. They both said that the HUMAN DNA that was detected was too degraded to show novel primate. That statement has been stuck in my head for many months with no resolution. There was something being said there and I just wasn't listening to it properly for some reason. The dialogue here and elsewhere has always been that there's no Sasquatch DNA in the GenBank to compare test results too. So does that mean we're simply stuck up a creek without a paddle? 

 

I don't think so because Dr. Disotell said the Human DNA was too degraded to show a novel primate. And folks that says it all if one thinks about it. It's also saying that what I've been saying equates to what Dr. Disotell said and I had simply missed the connection. What he had essentially told us then was that Human DNA that is NOT degraded COULD show a novel primate!!

 

I mean, what is the opposite of "Human DNA that's too degraded to show a novel primate"? It would have to be "Human DNA that isn't too degraded. And that's what I have been overlooking all this time!!! It reinforces everything I've been saying about not needing Sasquatch DNA in some genbank to discover the Bigfoot. Dr. Disotell had essentially already told us that without actually saying it.

 

I don't know if that has helped you at all, Twist, but it reinforces what I've said about primate DNA being primate DNA. Because if that's what we end up with after finding what we think is Human DNA then guess what........

 

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hiflier
44 minutes ago, NorthWind said:

Now what about soil? For example, finding prints like Madison and I do. No water in them, but still probably loaded with eDNA if they are what we believe they are. Can that soil just be collected in a little zip lock with a clean and sanitized tool?

 

16 minutes ago, BlackRockBigfoot said:

 

@hiflier any recommendations on soil sample collections?

 

 

I don't know enough about sampling soil to be of much help. Fecal material and burrows maybe? LOL. I do know that different soils will have a different effect on rates of degradation based on the microbial colonies each will or will not support as well as their abilities to hold water as well as an insect population. How much heat and sun as opposed to rain and shade. the ph of the soil and that's just for starters. Those variables coupled to a temporal influence on those variables plays a big role in DNA degradation rates. And I think moisture is the worst for soil DNA integrity. That said, maybe read this? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC140122/ I think soil samples are more costly because of the possible need to filter out other compounds that bind with the soil?

 

I know that with the Snellgrove DNA sample where there was thought to be Bigfoot blood and tissue on some screws in a board there was some confusion until the galvanizing that was on the screws was filtered out or neutralized somehow through some process. In a remote area, perhaps all one needs to be concerned with is the natural soil with no additives? And maybe if one is looking for a target species like primates? Although I'm sure that the process of metabarcoding (Disotell's method?) should show a primate anyway. I'm a bit lost trying to actually find a step-by-step a protocol for extracting soil samples, though.

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Twist
53 minutes ago, hiflier said:

I mean, what is the opposite of "Human DNA that's too degraded to show a novel primate"? It would have to be "Human DNA that isn't too degraded. And that's what I have been overlooking all this time!!!


If it were as simple as human DNA being to degraded. Then all other BF samples wouldn’t be tossed out as contamination.   I find it hard to believe that ppl of Disotells knowledge simply over looked this and I doubt that all Hilan Results were to degraded.  

Cant jump to conclusions here on the subject of DNA based on what was or wasn’t said by a scientist.   
 

Serious research needs to be done on the subjects of A) Can EDNA produce results that could show the right gene.   B.) Who and how can the gene be identified by. 
 

Both questions that should be answered before a grassroots movement has ppl spending money on collection kits.  

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hiflier
21 minutes ago, Twist said:


If it were as simple as human DNA being to degraded. Then all other BF samples wouldn’t be tossed out as contamination.   I find it hard to believe that ppl of Disotells knowledge simply over looked this and I doubt that all Hilan Results were to degraded.  

Cant jump to conclusions here on the subject of DNA based on what was or wasn’t said by a scientist.   
 

Serious research needs to be done on the subjects of A) Can EDNA produce results that could show the right gene.   B.) Who and how can the gene be identified by. 
 

Both questions that should be answered before a grassroots movement has ppl spending money on collection kits.  

 

Couldn't agree more on that. But if Human DNA only ever shows as Human, then why did Disotell say that the Human DNA was too degraded to show a novel primate? How could Human DNA, degraded or not, possibly show a novel primate? It doesn't leave a whole lot of room for interpretation. Too degraded to show novel primate? That makes no sense whatsoever. Someone has to ask "But, Dr. Disotell, what if the Human DNA wasn't as degraded as you said it was? If not, then would that mean there could be a chance of it showing a novel primate?" I wonder what his answer would be ;) 

 

@NorthWind and @BlackRockBigfoot Is this maybe what you are looking for? Soil Sampling.pdf 

 

Then you, me, and everyone need to know the general procedure that has to happen once samples are delivered to a lab. I also think this video shows why the soil samples  from the OP nesting site were so expensive to test:

 

 

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BlackRockBigfoot
1 minute ago, hiflier said:

 

Couldn't agree more on that. But if Human DNA only ever shows as Human, then why did Disotell say that the Human DNA was too degraded to show a novel primate? How could Human DNA, degraded or not, possibly show a novel primate? It doesn't leave a whole lot of room for interpretation. Too degraded to show novel primate? That makes no sense whatsoever. Someone has to ask "But, Dr. Disotell, what if the Human DNA wasn't as degraded as you said it was? If not, then would that mean there could be a chance of it showing a novel primate?" I wonder what his answer would be ;) 

 

@NorthWind and @BlackRockBigfoot Is this maybe what you are looking for? Soil Sampling.pdf 

 

Then you, me, and everyone need to know the general procedure that has to happen once samples are delivered to a lab. I also think this video shows why the soil samples  from the OP nesting site were so expensive to test:

 

 

Awesome.  I will check this out.

 

Thanks!

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NatFoot

@hiflier, regarding your last post - I think that's exactly what @Twist is saying needs to happen.

 

That question needs asked and answered before some grass roots project gets off the ground and people put a ton of effort into protocols, gear, etc.

 

Word games, and words do matter, won't work here. I know from previous posts you like to read into things (too much, IMO).

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