Sample 26 mtDNA Electropherograms Show Degradation/Contamination

Figure 1.  Ketchum S26 Electropherograms from SGP website.  Green bars above each peak proportional to quality of read.  Peak color indicates base:  A G  T  C.  For research and education only, "Fair Use." 

Melba Ketchum has consistently claimed in her paper [1], on radio, on TV, on the Sasquatch Genome Project (SGP) website [2], on YouTube [3],  and at every other opportunity,  that her samples are not contaminated or degraded, in spite of very anomalous results, many of which are easily explained by such.  She posted Sample 26 (the Smeja find) raw data mtDNA electropherogram files on the 
SGP website.  I downloaded all 272 such files, which require special software to open, examine and assemble into a complete mtDNA sequence.  I purchased DNABaser software from Heracle Biosoft to do just that.  It cost $159 and runs on my PC.  The above Fig. 1 is a screen grab of  six randomly selected, representative electropherograms of the 272.

This data was obtained by the Sanger Dideoxy Sequencing Method, outlined in Fig. 2.

Figure 2.  Sanger Dideoxy Method.  After Wikipedia.  For research and education only, "Fair Use."   

In this method, segments are elongated (as complementary strands) and terminated with "fluorochrom" (i.e. fluoroprobe, or fluorescent tag) nucleotides with different fluorescent wavelengths for each of the four nucleotides (A, G, T, C).  The successive elongated segments are separated by electrophoresis according to their length, then detected and identified by their characteristic fluorescent wavelength (stimulated by a laser).  Software makes the plots as represented schematically in Fig. 2 lower right or actually in Fig. 1 and generates a sequence (Fig. 1 below each electropherogram, Fig. 2 lower right).

All but the shortest (few hundred bp) sequences must be sequenced in segments, and then assembled by computer, based on overlaps, as shown in Fig. 3.

Figure 3.  Assembly.  Overlaps underlined.  After Wikipedia.  For research and education only, "Fair Use."


Mitochondrial DNA assembly is handily done on a PC.  Only very large data sets (e.g. whole nuclear genomes) from next generation sequencing, require a "super computer" or a network of linked servers to assemble.

Sanger sequencing and subsequent assembly does not work for severely degraded DNA, whether from a single or multiple sources.  This is because of the presence of sequence fragments which are self-primed and which are never the less elongated and tagged with a fluorescent probe.  These fragments coelute with the fragments of interest as shown in Fig. 1 ("Poor").  The "Excellent" electropherogram has no such coelution:  each peak stands alone and is separated from its neighbors so that the fluorescent detector can make an unambiguous base call (A, G, T, or C). The "Good" electropherogram can also be used because coeluting peaks are much smaller in most cases and can be ignored.  The few ambiguous, double peaks will hopefully be clarified by another overlapping sequence fragment.  Otherwise these few remain unknown bases (or possibly polymorphisms).  The "Poor" electropherograms are totally uninterpretable.  They have multiple overlapping peaks of comparable intensity.  Those "?" electropherograms might have some salvageable sequence runs, but should be considered dubious at best.   So, two usable e-grams out of these six.  A well collected and preserved single species DNA sample would produce only usable e-grams.

Unfortunately, the 272 electropherograms contained only 47 that could be assembled into a sequence of 5260 bp out of an expected 16,568 bp human mitochondrial genome.  The remaining 225 electropherograms contained only 11 which could be assembled into a 1923 bp sequence.  Other assemblies were shorter still.

The first assembly (47 e-grams) aligned with the published S26 mtDNA sequence [4], base positions 11401-16551, only 96% with 74 gaps.   The second best assembly (11 e-grams) aligned with published [4] S26 base positions 13718 - 15435 only 97% with 27 gaps.  This is rather poor agreement between these raw data and the published S26 sequence [4].   The 47 e-gram sequence aligned with the 11 e-gram sequence only 93% with 1643 identities and 69 gaps.

Thus, only 32% of the mtDNA genome could be sequenced with these data, and the sequence agreed very poorly with the published S26 sequence produced by Family Tree DNA [4].  FTDNA assumes a human sequence and uses human primers, so DNA of any other species present would not be sequenced. 

These data call into question the mtDNA sequencing and haplogroup designation of Sample 26, and hence also the Ketchum claim that this sample represents a human-unknown primate hybrid.  Most likely, the sample is a black bear contaminated by one or more humans and severely degraded, as shown in my previous blogs. 

References

[1]  Ketchum M. S. et al. (2013) Novel North American hominins: next generation sequencing of three whole genomes and associated studies. DeNovo 1:1.  Online only: http://sasquatchgenomeproject.org/ 

[2] http://sasquatchgenomeproject.org/sasquatch_genome_project_002.htm

[3]  https://youtu.be/yW4yPPXPfSw?t=1

[4]  http://sasquatchgenomeproject.org/linked/supplemental-data-2-mito-mutation-chart-complete-final.pdf

My Response to Melba Ketchum’s Facebook Post About Me

In  response to Chris Noël’s comment on Melba’s Ketchum’s FB page that I was “slamming” her sasquatch conclusions on the Internet  (to which I totally confess) , Melba Ketchum posted the following on her FB page:

 “1. His degree is not in a discipline that allows him to be qualified to review the paper. 2. He didn't use bioinformatics software which has to be done in order to evaluate the data.
3. You can't use only statistics to evaluate the sequences nor by tearing it down into little sequences unless you have software/expertise to do so.
4. He early on discounted the mtDNA findings using the wrong reference sequence. 5. He then changed his mind and said the mtDNA human sequences were correct 6. He reviewed the paper and said our nuclear DNA findings were correct and told me I could post his positive review (I have it in writing). However, I didn't post it because he is not qualified to review the paper and he even stated so in his correspondence. Now, he has changed his mind again and says the nuDNA is incorrect but he's not acknowledging that he's not qualified to review the manuscript. I have generally just ignored him for all of these reasons.”

I post my response here, since she defriended me on FB quite some time ago:    

The first 6 comments correspond to her numbered comments above.

1. Melba’s degree is in veterinary medicine, NOT genetics or anthropology, or even biochemistry.  By her criterion she is not qualified to write the paper she did, nor are ANY of her coauthors.  Many people change their professional orientations/interests.  Melba has no degree in forensic science, yet testifies in court on her DNA findings, in capital cases.   My Nobel Prize winning PhD thesis advisor said he never decided between biology, chemistry, or physics.  He got the Nobel Prize in chemistry for his interdisciplinary work.  Any respectable chemist understands DNA enough to do what I did.  It’s a chemical compound, or more accurately a family of related compounds, differing by mutations of the bases.    

2.  I used the same bioinformatics software, BLAST™ from NCBI, that she did.  It doesn’t take special software to tear a sequence down into little 60 base pair segments as HER colleagues did.  The analysis can be done on Microsoft® Excel. 

3.  My paper did not involve tearing down a nice long sequence into little bits, loosing information as you do so.  That was her colleagues’ approach, which experts at the NCBI said   “makes no sense.”   I did this as a side exercise to compare results to hers.  I am confident that her team never tried to align S26 against a bear or S140 against a dog and compare those to the human alignments.  I did, and the alignments, by both her method and mine (which searches the whole sequence), favor bear and dog, respectively, over human or any other animal.  I have used numerous databases throughout my career and hold two US patents for inventing databases  (US 6,732,054 and US 7,532,785) as explained in the Preface to my first paper.

4.  Her Supplementary Data 2 was poorly constructed with no headings, footnotes, or text explanation.  It took me awhile to realize that there are two types of data in it: HVR-1 only and full sequences.  This should have been explained in her paper.  There are even two sequences off to the lower right that are not identified in table or text, labeled, or aligned with the rest in columns.  Some mutations have no suffix (A, G, T, C).  She took some raw output and never even bothered to critically examine it or make it intelligible to a reader.  “Supplementary” does not mean unexplained or unformatted.

As an example of how confusing her Supplementary Data 2 is, read the following from an experienced genealogist, A. John Marsh.  Melba had, of course, posted on FB his conclusions on the T2b samples as evidence of support.  He was totally confused by her very sloppy reporting in Supplementary Data 2 and had to retract his posts.  I guess I can be excused for changing my mind too.

·         From: Alister John Marsh <ajmarsh@arrrg.org>
Subject: Re: More on Bigfoot.
To: "Haskell Hart" <hvhart@swbell.net>
Date: Sunday, March 24, 2013, 9:52 PM

·         Hi Haskell,

·         See my earlier reply to you where I noted that I had made a mistake, in that some sequences were using rCRS as the reference, and some were using RSRS.  Family Tree DNA did the mtDNA sequencing, but they apparently did it a year or more ago when they used the rCRS reference, but now they use the RSRS reference.

·         When I get back from a trip on Friday, I will have a fresh look at T2b.

·         Regards,

·         John

Bottom of Form

Later he wrote to me:

“I would rather you did not quote me on anything to do with T2b Bigfoots until I can reevaluate things.  I have made a posting on the genealogy-dna list confessing my error.”

NOTE:  I never heard back from John, nor have I seen any more of his posts on Melba’s FB.  Since this exchange I have shown that Samples 2(T2b), and 39b(T2) are outside the range of human mutations, Sample 12 (T2b) is marginal at 2.3% chance of occurring, and only Samples 1 (T2b) and 36 (T2b) are from a human population.  These and related results are explained in my second paper.

AGE OF T2b:

Web site
http://www.nature.com/srep/2012/121018/srep00745/full/srep00745.html
If T2b is 12-10kya, and T1/ T2 coalesced about 19kya, T2b might very roughly originate about 10,000 to 15,000 years ago.

THINGS I NOTED ABOUT MUTATIONS DIFFERENT IN BIGFOOT AND HUMANS:

One thing I noted was that all the 52 number diverse T2b haplogroup listed humans in the T2 project had a mutation 146T, but none of the Bigfoot had that mutation. It seems in fact that all T haplogroup have 146T. I am guessing that the earliest common ancestor of all Bigfoots had a back mutation on that marker to the CRS value.

Another thing I noticed was that all Bigfoots which appear to have been tested on the lower number markers, have mutation 73G. Yet not one of the
52 human mtDNA T2b persons had the mutation 73G. Why not? Was 73G a very early mutation in the Bigfoot line?

All the fully tested 4 Bigfoots had the 263G mutation, but not one single one of the 52 humans had 263G. Why not?

It seems all human T2bs have have 16187C and 16189T, but no bigfoots have either. In fact, all human haplgroup T are postive for both these mutations, so presumably in the common ancestor of all Bigfoots there was a mutation reverting to CRS.

According to the Ketchum knockers, all the mtDNA Haplotypes in her project are modern contamination. All of these Bigfoot haplotypes are different.
Isn't it a bit puzzling that all of these humans mistaken as Bigfoot have different T2 mtDNA haplotypes, all have 73G and 263G mutations not found in humans, and all seem to have had back mutations on 146T, 16187C and 16189T, where these back mutations are apparently not found in any human T2b s?


John.Along with the fact that all Bigfoots seem to have several different mtDNA mutations from each other, they also are found in 5 different states. It suggests that if a single T2b human female mated with a Bigfoot male 13,000 years ago, that the descendants of the T2b ancestor have spread widely in USA since then.

AGE OF T2b:

Web site
f T2b is 12-10kya, and T1/ T2 coalesced about 19kya, T2b might very roughly originate about 10,000 to 15,000 years ago.

THINGS I NOTED ABOUT MUTATIONS DIFFERENT IN BIGFOOT AND HUMANS:

One thing I noted was that all the 52 number diverse T2b haplogroup listed humans in the T2 project had a mutation 146T, but none of the Bigfoot had that mutation. It seems in fact that all T haplogroup have 146T. I am guessing that the earliest common ancestor of all Bigfoots had a back mutation on that marker to the CRS value.

Another thing I noticed was that all Bigfoots which appear to have been tested on the lower number markers, have mutation 73G. Yet not one of the
52 human mtDNA T2b persons had the mutation 73G. Why not? Was 73G a very early mutation in the Bigfoot line?

All the fully tested 4 Bigfoots had the 263G mutation, but not one single one of the 52 humans had 263G. Why not?

It seems all human T2bs have have 16187C and 16189T, but no bigfoots have either. In fact, all human haplgroup T are postive for both these mutations, so presumably in the common ancestor of all Bigfoots there was a mutation reverting to CRS.

According to the Ketchum knockers, all the mtDNA Haplotypes in her project are modern contamination. All of these Bigfoot haplotypes are different.
Isn't it a bit puzzling that all of these humans mistaken as Bigfoot have different T2 mtDNA haplotypes, all have 73G and 263G mutations not found in humans, and all seem to have had back mutations on 146T, 16187C and 16189T, where these back mutations are apparently not found in any human T2b s?


John.

*****************************************************************************

5.  I pointed out mtDNA anomalies to her early on in my first review.  I have published on the Internet a full analysis of these sequences in my second paper.  Many are anomalous to the point that they do not match any of the 27,156 complete human mtDNA sequences in the NCBI Nucleotide database.  Nor do these fit into the mtDNA PhyloTree of accepted human haplogroups.  Eight of 18 samples with complete sequences have less than 1% chance of belonging to the human population, contrary to a position she has steadfastly maintained for all of her mtDNA samples.  This is further explained in my second paper.

6.   She (Robin, her publicist) DID ask for and received permission to post my comments, which she did on Melba’s FB page.  She misspelled my name and ADDED “Way to go Melba Ketchum” without my permission.  See my previous blog.

 7.  I shared my results at every turn with Melba, hoping for some constructive feedback, but I got none.  In her last note to me she referred to it as “nastiness” without explanation.  I have since ceased direct communication.   Before this she called me and we talked for about an hour on general subjects until she finally asked me not to publish my first paper that is now on the Internet.  Her only reason was that she had access to much more data (four terabytes) and it all pointed to the existence of sasquatch.  Apparently most of it did not appear in her paper.   I did receive some of her colleagues’ results and conclusions on her sequences through her. She asked me not to share these, and I haven’t.  They wish to remain anonymous (One might wonder why.).  She unfriended me on FB, so I cannot respond to her there.  Science does not do “anonymous.”  These coworkers should have been mentioned in her paper as should their database search results. 

8.  I clearly indicated my background in the preface to my paper.  People can judge for themselves whether it is adequate to compare a string of only four possible letters: A,T,G,C (with some rare minor variations).  This is all I ever did.  I accepted and used her published results (the sequences) without comment about how they were produced.

9.  I have shared my first paper with two renowned geneticists/anthropologists familiar with her work, and both have made positive comments.  Also, both of the anonymous reviewers made positive comments about my methodology when I attempted to publish my paper in an online journal (their comments are in the Preface to my first paper).   The reason it wasn’t published was because the editor took the position of one reviewer that it was essentially based on her unacceptable results and conclusions:

“There is no scientific evidence that any other hominids currently live. The only reference is to a paper in a journal that has only one edition. If there was any evidence then a publication such as Nature is the more likely journal in which this would be published.”

10.  Changing one’s mind is scientifically in order as long as reasons are explained openly and honestly, as in my paper.  Clinging to conclusions that have been shown to be unsupported by one’s own results while not critically answering the challenge is unscientific.  Real scientists debate issues openly.  They don’t “just ignore” them.  Reasonable people can disagree, but scientists base their disagreement on their own work AND critical analysis of opposing views.  The latter has as yet not been heard from Ketchum.  

11.  On one website alone, nearly 100 people have downloaded my first paper.  A few have asked questions or made positive comments.  NO ONE has commented negatively or pointed to any error.  The topic of the Ketchum paper had previously attracted thousands of comments there, positive and negative. Since I posted my paper it’s been close to dead silence.  I welcome critical comments and have said so. Melba doesn’t.