The Sasquatch Genome Project: A Failed DNA Study

[hvhart]

hiflier,  when a species is unknown, a universal primer is the way to go, at least until you know what it is similar to.  If you use a species specific primer as suggested, and if you  miss the mark it may not amplify and sequence if it is sufficiently different.  Hold off on NOTCH2NL until you know what you are dealing with.  You will need much more sample for a nuclear gene like this than for a mtDNA gene.  A few hairs will not do.  The contract lab may just want to do what they are set up for.  This is not a routine job.  If they use they wrong primer you get nothing.  A universal primer will tell you much more at the early stage of an identification.  Ketchum made the mistake of using human primers for nDNA genes and microsatellite loci and got anomalous results or in some cases no results.  None of this will work with eDNA because the sequences are too short and may not even cover the area of interest or the primer region (unless you are very, very lucky).  BTW I would suggest Mitotyping Technologies, the lab that analyzed Sykes' samples.  Ask for Terri Melton, a coauthor.

[NCBFr]

5 hours ago, hvhart said:

 

This is a very good question.  If a universal mammalian primers are used for the HV1 region, cytochrome b, or cytochrome c oxidase 1, you should get the corresponding sequence for these mtDNA regions.  Every effort must be made to decontaminate the sample first, e.g. a hair should be ultrasonicated in water, not vortexed in alcohol/water as Ketchum et al. did.  The sequence can then be compared in BLAST(R) to known sequences AND a phylotree of hits should be constructed (Chapter 7).  Perhaps it matches no species close enough for a species ID, however, its position in the phylotree of hits will tell you what it is most closely related to., human, chimp, other primates, etc.  I did this for Ketchum et al. Samples 26 and 140, and these were in exactly the right places for a black bear and a dog, respectively (Figs. 12, 13 and 14).  Obviously a reference sequence from a sasquatch body part is the "gold standard" here against which all subsequent samples can be compared.  In its absence, however, a phylotree can tell you a lot if you have a pure species sample and use the proper primers.  Researchers who present evidence for a new species make phylotrees from DNA sequences of related species plus their "newbie."  A good example is the Lesula monkey, https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0044271 .  Here the researchers had the new species in hand, however, which is a distinct advantage.  They also presented lots of other evidence for a new species.  Compare this to the ridiculous phylotrees of Ketchum et al.  in Figs 16 and 17, (Chapter 7).  Also, extinct mammals (cave bear, short-faced bear, cave lion and others) plus Denosovans and Neanderthals (a complete genome) were sequenced from small bone fragments amid many contaminants, especially microbes.  Keep reading.  I'm here to help you through it if necessary.    

 

Thank you for the thorough answer.  Clearly I got some work to do before I can respond with follow-ups (up to p. 21). 

 

PS - I have always been a little in awe of DNA and how it works.  However, as I really start to understand it I have to wonder how it came to being.  It is almost too perfect.

[hvhart]

1 hour ago, NCBFr said:

 

Thank you for the thorough answer.  Clearly I got some work to do before I can respond with follow-ups (up to p. 21). 

 

PS - I have always been a little in awe of DNA and how it works.  However, as I really start to understand it I have to wonder how it came to being.  It is almost too perfect.

You made my day.  Keep it up!  I was in awe of chemistry as a HS student.  Just keep learning and don't be afraid of something because it is difficult to understand.  It just takes time and effort.  At 77 y.o. I am running out of both.

[hiflier]

15 hours ago, hvhart said:

Ketchum made the mistake of using human primers for nDNA genes and microsatellite loci and got anomalous results or in some cases no results.

 

Of course! I should have kept that in mind since your book described how that approach was such a failure. So a universal primer to get inside the ballpark and THEN narrow down the research. Got it. Thank you.