Monday, September 16, 2013

EXCLUSIVE - Newly leaked Information shows that the Ketchum Bigfoot DNA Study PASSED PEER REVIEW - UPADTED -09/18/2013

This is Dr. Kethcum's Response to the leaked information I received below from her FaceBook Page:


I am so upset that the reviews got leaked. That means I was once again betrayed by an insider. On the up side, I feel a huge relief about it. Now I don't have to sit on this horrible secret anymore. The world now knows how unethically the scientific has behaved. It also knows that we DID pass peer review and with reviews that came from geneticists that work with whole genomes. At least that is what I was told.

Dr. Ketchum verifies the autheticity of the Peer Reviews via a post on her FaceBook page:

As far as all of the documents that were leaked, they are authentic. I didn't leak them though. Some submitters as well as others associated with the project had access to this information so I don't know who did. Those communications are not supposed to be leaked. So if anyone gets sued, it won't be me. They can subpoena the blogger's email trail. I actually am happy about the leak though. At least now everything I have said can be authenticated including the ridiculous and biased nature of the reviews. Now people will know the truth and that we did pass peer review
Dr. Ketchum's DNA Study  the "Novel North American Hominins, Next Generation Sequencing of Three Whole Genomes and Associated Studies" did pass peer review in January 2013. The journal then known as the "Journal of Advanced Zoological Exploration in Zoology" (JAMEZ) gave the Ketchum DNA Study passing reviews and was planning to publish the study in its inaugural edition on or about January 11th, 2013.

Late in the evening of Wednesday January 9th, 2013 I received a phone call from David Paulides, Director of North American Bigfoot Search (NABS) the primary partner of the DNA Study. David informed me that he had just hung up the phone with Dr. Ketchum and she had been given notice that the DNA paper had passed peer review with JAMEZ and the publishing date "would be on or about Friday, January 11th, 2013". David said we should be prepared for some moderate media attention and possible interview request.



Then inexplicably the next day, January 10th, 2013, I receive the following email from David Paulides:


Apparently  someone had gotten to the staff of the JAMEZ and intimidated them so severely that they were now refusing to publish the paper. Even the attorney for the JAMEZ was under so much pressure from some outside source that he had threatened to resign if the JAMEZ published the DNA study. (Editors Note: The above information was previously disclosed by Dr. Ketchum during radio interviews in February and March of 2013. This email is provided as independent collaboration of her claims made during these interviews)

To prove the journal did exist below is a screen shot  of the JAMEZ's "Calls for Papers".



Later Dr. Ketchum acquired JAMEZ in order to preserve the passing per reviews. The JAMEZ was renamed  Denovo (http://www.advancedsciencefoundation.org/). Dr. Ketchum then published the "Novel North American Hominins, Next Generation Sequencing of Three Whole Genomes and Associated Studies". Critics blasted her for doing this and claimed the paper never passed peer review. Due to confidentiality rules Dr. Ketchum could not make public the passing peer reviews from JAMEZ. Many critics, knowing these rules, used this against her and mounted an unprecedented personal smear campaign to discredit her and the DNA Study.

On Sunday, September 15th, 2013 I received a Facebook friend request from an individual I did not know. But since we had several "Facebook mutual friends" in common I accepted the friend request and did not think anything else about it.  Late yesterday afternoon I received a Facebook message from this individual telling me he wanted my email address. He expressed his extreme dismay at the treatment I had received in the group "Bigfoot Community" the previous two days (http://www.bigfootevidence.blogspot.com/2013/09/ex-publicist-sally-ramey-opens-up.html) and was tired of the treatment I and the other supporters of Ketchum DNA Study had received over the past few months. He told me that he had inside information that would "Blow the lid off this thing and shut the critics up". He would not tell me what he had, he said "you will understand when you get my email". He also made me swear that I would keep his identity secret because the information he was giving me was "extremely sensitive". I gave him my email address and waited.

On Monday, September 16th I received his email. It contained a screenshot of an email from Scholastica, the publishing house for JAMEZ. Along with this screen shot I received the JAMEZ Peer Review with the authors' (Dr. Ketchum) responses and the Journal Nature's Peer Review from the second submission with authors' responses.

The email print screen that I received from the anonymous source clearly states that the Ketchum DNA Paper - "Novel North American Hominins, Next Generation Sequencing of Three Whole Genomes and Associated Studies" DID PASS A INDEPENDENT PEER REVIEW FROM A SCIENTIFIC JOURNAL. The status for the manuscript read: "ACCEPTED for publication with revisions" Below is the screen shot of the email:





Below is the JAMEZ peer review with Dr. Ketchum's (authors') responses that I received from the anonymous source. This is the peer review that was accepted for publication:

Authors’ Response to Review

Referee A
I appreciate having had the opportunity to review this interesting investigation into such a controversial subject and recognize the enormous constraints under which the authors and their collaborators have labored.
Major concerns with this manuscript:
1. A difficult two-part thesis is posed that is inadequately substantiated by the analysis presented in the manuscript. Both parts (i.e. Part 1 - previously uncharacterized hominins exist in North America; Part 2 said hominins are the descendants of a putative hybridization event involving an ancient uncharacterized hominin and a modern human). A thesis this complex and counterintuitive requires significantly more in-depth analysis and consideration and should be developed through a series of peer-reviewed publications. Add in the idea that more than one species of hominin may be present in North America and the effort to make a convincing case is multiplied.  

Author’s Response:
We removed the discussion of the different variants of Sasquatch living in North America simplifying the manuscript and added an alternative hypothesis outside of hybridization to the discussion so further discussion of their origins can be fully developed in future manuscripts. Lines 688-696.

 2. The presentation of the thesis is overburdened in three ways: a) the considerable inclusion of unsubstantiated eyewitness accounts and folkloric/pop-cultural ideations and their presentation as foundational facts that validate the analytical data; b) the presentation of a series of anomalous results generated using routine methodologies resulting in a sub-narrative that is curious and interesting, but unnecessary given the fact that cutting edge technology has been employed to generate sequence data sets that should allow for incredibly detailed analyses yet lead to relatively unambiguous conclusions; and c) the results from Next Generation Sequencing methodologies, especially related to the analysis of the nuclear genome are fairly superficially treated so as to be unclear whether the authors have selectively aggregated sequences to support a favorable placement among primates. As stated in Perleman et al. (2011; PLoS Genet 7(3): e1001342. doi:10.1371/journal.pgen.1001342, "...primate taxonomy is both complex and controversial, with marginal unifying consensus of the evolutionary hierarchy of extant primate species," and given the expertise of one of the authors in this particular area, it is a bit astonishing that a more in-depth and stepwise treatment is not provided. The decades of work published by the Goodman lab provide an excellent roadmap for a convincing analysis. In summary, this work would achieve a much higher degree of analytical credibility by a) limiting folkloric/cultural references to the minimum necessary, b) moving many of the references to anomalous results to a supplementary role (2nd publication, perhaps), c) fully leveraging the treasure trove of information yielded by the Next Generation Sequencing.

Author’s Response:
a)     The introduction with popular information was requested by another reviewer.  Since there is broad interest in this manuscript, that reviewer felt that the inclusion of such information was important.  I will leave it up to the editor whether this should be included or not.  It should be noted that 2 of the authors of this paper did have eyewitness encounters at the invitation of people that have interaction with this species so it is not entirely unsubstantiated. 
b)     The previous results were left in the manuscript because they supported the next generation sequencing results and also because they were obtained from a large number of purported Sasquatch samples, which behaved in the same manner as the three samples utilized for Next Generation Sequencing.
c)     The purpose of the manuscript was to prove that the Sasquatch exist.  We chose chromosome 11 as our starting point for building our supercontig in order to generate the phylogenetic tree since the scope of the manuscript was to determine whether a novel primate did exist in North America.  This was the logical way to proceed and with the length of the contigs and also with more than 379 genes analyzed (for another manuscript in the future) we feel that this was the direction to go to prove that they exist and as a preliminary study.  In the beginning of the study, we had already ruled out non-primate species with not only the mtDNA findings but also with PP16 since it only gives results for ape and human with the exception of the 103 peak at Amelogenin that appears when there is animal DNA present in a sample.  The 103 peak was not found in any of the purported Sasquatch samples that were screened using PP16. Furthermore, fully leveraging the genomic data will take years.  This manuscript is just the beginning of the study.
3. A significant amount of work leading to the generation of the data central to the thesis was outsourced. It is very difficult to manage the quality control and release standards of contract laboratories from the outside. This does not automatically call such data into question. However, when coupled with a significant amount of anomalous and ambiguous data, such concerns must be considered. This would be especially true concerning Next Generation Sequencing of unknowns where heterologous libraries may result from contamination. This technology is much more sensitive to the detection of contaminating gene sequences than others, and the possibility that contaminating sequences may affect the development of a consensus sequence should be given considerable attention. Reference bias should also be given consideration in the development of a consensus sequence. Similar work typified by that of Pruefer et al. (Nature, 2012/06/13/online) could provide good guidance concerning appropriate quality control analysis.

Author’s Response:
The outsourced laboratories were accredited and work with human samples, which require regulation.  We have correspondence where the laboratories were confused about the results and in some cases doubled checked their findings.  Furthermore, more than one laboratory repeated the analysis at least to some degree supporting the data of other laboratories.  They all utilized the same extractions that had been aliquoted.  As far as the Next Generation data, it confirmed the mitochondrial findings from previous laboratories as well as novel sequence.  That is why it was important to leave the original data in the manuscript.  Furthermore, according to supervisors in data analysis at Illumina, the Q30 quality scores would have been remarkably lower if contamination had been present in the genomes due to competition by the various sequences.  That also would have affected the mitochondrial findings which were consistent with the original mtDNA sequencing.
4. Conflicting statements occur concerning the validation against submitter contamination:
"Control DNA was obtained from the majority of the submitters and was profiled using Promega PowerPlex® 16 (20). All submitters yielded complete profiles."

Author’s Response:
This has been corrected to clarify on line 200: All submitters “tested”

At the very least the STR profiles for the submitters of Samples 26, 31, and 140 should be presented, as these samples yielded the most significant data of the study.
Author’s Response:
Since the submitters’ names are given per their request contractually in Table 1, it would be inappropriate and unethical to publish their DNA profiles as it would link to their identity. However, we have them on file.

5. Sample 26 - It is stated in the study that Sample 26 was derived from a shooting incident. The inclusion of such a sample in the study may be inconsistent with contemporary scientific ethics concerning the treatment of both human and animal study subjects and the procurement of research specimens from such. Moreover, this description casts the provenance of such a sample as murky, at best.

Author’s Response:
We have removed any references to the alleged shooting incident. Line 481

6. The bioinformatics should include gene sequences from expected outlier species that may also be capable of contributing contaminating nucleic acids. For example, a BLASTN search using Sample 26 does turn up some exceptionally strong homology with a gene from Ursus americanus (DQ240386.1). This would support the idea that the consensus sequence may have been affected by contaminant sequences.

Author’s Response:
There will always be some homology with other species when short random sequences are chosen, however, your example of bear contamination can be completely ruled out considering none of the laboratories handling the samples have bear samples.  Furthermore, forensic screening would have given a 103 peak at amelogenin on PP16 should there have been non-primate contamination.  Sequencing of the mitochondrial DNA with universal primers also would have shown any contamination of the original extractions with non-human DNA.  Additionally, that is a single isolate from the black bear 7193328 brain-derived neurotrophic factor. Not only is it preset in Sample 26, but it is just as present in Sample 140.  Furthermore, DQ240386.1 is 489 bases. I wonder why such a small sequence, ie 489 bases out of 2.7 million bases was the focus of this critique.  DQ240386 is statistically significantly aligned with primates and carnivores.  In fact, BLASTing DQ240386- ring tailed cats of the raccoon family and seal have as much alignment as Ursus americanus. The maximum score for raccoon and seal are about 850. Maximum score for Ursus americanus is about 900. Max score for Sample 26 is 538. This shows contamination bias.

Minor concern
1. The use of “hominin” in the introduction, as opposed to hominid, creates the perception of bias toward folkloric and unverified eyewitness accounts that seem to emphasize human characteristics. The general discussion of such creatures tends to split along two lines: human or human-like and ape or ape-like. The term hominid, as related to the Family Hominidae, is the higher order, and, thus, the broader grouping. Visual description from a distance might allow for a reasonable description of a hominid, but close examination of the organism or its remnants is required for its placement into the Hominini. Thus, to establish initial objectivity, the authors should use a qualifying adjective such as “putative.”

Author’s Response:
Pan/Homo divergence occurred between 5.4 and 6.3 million years ago (refs below). Since the mtDNA speciation is not only unequivocally modern human, but can be dated by the haplotypes obtained in this study to as late as 13,000  to 26,000 years ago, these individuals must be included as hominins.
We did add the word “putative” in line 85.


Bradley, B.J. (2006). "Reconstructing Phylogenies and Phenotypes: A Molecular View of Human Evolution". Journal of Anatomy 212 (4): 337-353. doi:10.1111/j.1469-7580.2007.00840.x.
Wood and Richmond.; Richmond, BG (2000). "Human evolution: taxonomy and paleobiology". Journal of Anatomy 197: 19–60. doi:10.1046/j.1469-7580.2000.19710019.x. PMC 1468107. PMID 10999270.

Referee B
1.       This is clearly important information that I hope the public will have access to soon. However, I was immediately taken by surprise after thoroughly reading the manuscript to see such a high reference to Hominins, including the title. I was surprised because there is no substantial evidence presented by the author that the species identified in the 3 whole genomes is a biped. Eye witness accounts are the only data presented to determine or substantiate a biped of any kind. It would be much more appropriate to delineate in this manuscript that novel genomic evidence highly suggests an unknown species living contemporaneously in the Continental and Subarctic United States and Canada within the Order: Primate. There is no conclusive evidence that the unusual data found in the follicular hair morphology or the tremendous genome results indicates a "hominin". The tribe Hominini (of Homininae) is reserved for Homo, post Panini divergence. Genetic information alone is not enough to classify the species within a tribe or clad under order - Primate. Though there is substantial alignment in some data relating to hair and other DNA studies, it is too presumptuous to purport the extant data included in the manuscript conclusively classifies this unknown species as hominin. An example (to the author) of how to, perhaps, revise:
---The genomic data of sample 26 indicates the specimen allegedly collected from an unknown creature is certainly novel and indicates an unknown species. The phylogenetic trees of sample 26 indicate a species highly aligned with the order: Primate. If the alleged story related to us by the hunter who submitted the sample, the species is closely related to the hominin tribe because the hunter claimed to have seen the creature walking "upright". Our chromosomal analysis would substantiate a close hominini relationship. Further study and analysis will be ongoing to determine such classification. The phylogenetic trees are quite compelling and are probably the most substantial of the information given within the manuscript. However, the information and data is not satisfactory to hypothesize anything more than a living unknown primate. The molecular genetic data is quite compelling and enough to publish in and of itself. But in order to use taxonomic language in this manuscript, especially related directly to the species itself- is inappropriate. In order to include the level of specific taxonomy presented in the manuscript such as hominini, gross anatomical evidence along with molecular physiological evidence must be included in the manuscript. Even a clear video of this species actually walking bipedal would give some sort of gross anatomical evidence. But eye witness accounts are not enough to make such hypotheses. I do, however, believe the eye witness accounts are important and should not be excluded. They are just not appropriate to utilize for evidence to propose any hint of taxonomy, including hominini.

Author’s Response:
Pan/Homo divergence occurred between 5.4 and 6.3 million years ago. Since the mtDNA speciation is not only unequivocally modern human in 100% of the samples included in this study, and furthermore, can be dated by the haplotypes obtained in this study to as late as 13,000 to 26,000 years ago including the mtDNA from the 3 Next Generation genomes, these individuals must be included as hominins.  The human mitochondrial DNA haplotypes were consistent between the Next Generation Sequencing and the original mtDNA whole genomes sequenced in the beginning of this study.


1.     Bradley, B.J. (2006). "Reconstructing Phylogenies and Phenotypes: A Molecular View of Human Evolution". Journal of Anatomy 212 (4): 337-353. doi:10.1111/j.1469-7580.2007.00840.x.
2.     Wood and Richmond.; Richmond, BG (2000). "Human evolution: taxonomy and paleobiology". Journal of Anatomy 197: 19–60. doi:10.1046/j.1469-7580.2000.19710019.x. PMC 1468107. PMID 10999270.


2.         The molecular genetics in this manuscript are the most important and it would be important to include information regarding the analysis of the whole genomes. The phylogenetic trees are exciting to look at and speculate about, but there is not enough analysis to determine phylogenicity. I highly suggest including language that 'it is recognized that continued analysis of the nuclear DNA will be required in order to determine phylogenicity. The genomic information is certainly impressive, but not as conclusive as the manuscript proposes. It is inappropriate to make connections between the various samples. In fact, I believe this weakens the manuscript due to the reality that the genomic data and mtDNA analysis does not align well against each other (except for 2 of the whole genomic sequences). There are similarities, and those similarities are important to note. But conclusions cannot be drawn about the inter-relatedness of these various samples. The number of samples do indicate high alignment with Homo along chrm11. However, there are many other novel sequences across the several samples that creates confusion for the reader. Perhaps it may be helpful to split the manuscript up into several different studies- or simply divide the manuscript up into major sections to delineate the different evidenciary aspects. But to attempt to make links between the hair samples and the novel genomic sequences and other DNA evidence is premature. Tremendous work would need to take place with substantial evidence, including gross anatomical evidence, to make such linkages between the various samples in the study.

Author’s Response:
Mitochondrial DNA has been universally utilized to determine species by the scientific community.  Since the mtDNA in Sasquatch is not only unequivocally modern human in 100% of the samples included in this study, that places the Sasquatch as human.  However, since the nuDNA is novel to a large extent, the speciation of a potential progenitor is what is in question, especially since there are gene sequences that align 100% with human interspersed in the nuclear genome.

3.         The other major problem with this manuscript is, again, the alleged specimens these samples came from. It is important to state the samples came from alleged Sasquatch specimens, which is done well throughout the manuscript, but not consistently. It would be helpful, however, to include additional evidence to corroborate the theory that the samples came from a large non-human biped. Video evidence of this species walking bipedal would be important to help strengthen the manuscript. In addition, it is inappropriate to make statements regarding the dynamic between Homo Sapien DNA and the whole genome sequence. It is appropriate to include statements about distant relationships with the order- Primate. But it is certainly premature and the evidence is not conclusive enough to make such close connections between this unknown species and Homo Sapiens. I do believe that there is strong data in the whole genomes to suggest alignment with Homo Sapiens. However, there is not enough evidence to make statements about hybridization events. Hybrid phenomenon across the Homo/Pan/Gorilla taxonomy are currently highly debated in the scientific community. Making such claims is far too premature for a manuscript like this. The manuscript demonstrates a universal bias toward a hominini hypothesis. This anthropomorphic bias is problematic for this manuscript. A scientific manuscript is intended to present facts, not biases. If the editorial board decides to publish this manuscript with such a clearly "hominid" favoured bias, they certainly should proceed, but I recommend doing so with a strong disclaimer that an authored bias does exist in the manuscript.

Author’s Response:
Since the mitochondrial DNA places them as human, in both the original sequencing of the mitochondrial whole genomes as well as the Next Generation Sequencing, it is not premature to align them with Homo.  We did add another hypothesis in addition to the hybridization theory on lines 692-696. In consideration of the mitochondrial whole genomes, these are the only two viable hypotheses available.


5.    The inclusion of the Q30 scores was very important for the author(s) to do. This data strengthens the manuscript more than anything else included (if editors wanted to publish based on the Q30 scores alone, then there would be no reason not to publish). Furthermore, I am also most impressed with the presentation of the phylogenetic trees for the 3 whole genomes. They clearly identified primate relationships and alignment- though sample 31 seems to be very different from the other two whole genomes. That is concerning and confusing. It may make more sense to remove sample 31 altogether until the study can further understand and explain the huge difference with sample 31 (including conclusive evidence, not speculation). I am also pleased with the inclusion of the high definition pictures of human hair morphology versus the unknown species comparison. However, it would strengthen the manuscript to cross reference human morphology so as to substantiate the statements regarding the comparisons. I am also pleased to see the substantial amount of data and work taken to ensure high quality DNA samples with clear and appropriate techniques

Author’s Response:
References 15-19 refer to hair analysis and human hair morphology as well as animal hair analysis. As far as Sample 31, it should be included because it has the same characteristics as all of the other samples, human mitochondrial DNA and novel primate sequence in the genome as well as human sequence.  In the original manuscript we discussed that eyewitness accounts clearly note physical differences related to geographic location in North America.  We feel that 31 is such a variant.  We had taken this information out of the manuscript but will re-introduce it if requested.  The fact that overall, the genome is consistent with the others as far as its makeup clearly establishes its place in the manuscript. 
6.    The pictures included thus far of a supposed creature that is claimed to be a Sasquatch is of terrible quality and should be replaced with much more clear photographic evidence or video. These pictures weaken this manuscript. The inclusion of an image of sticks being put together in some kind of pyramid is inappropriate. I would strongly suggest removal of this image. 

Author’s Response:
We included the stick structure because one of the samples (168) was obtained within this structure.  Not only did this document chain of custody for this sample but the fact that a viable sample was found within the structure supports the discussion of eyewitnesses encountering unusual stick structures within areas purported to be inhabited by the Sasquatch.  As far as video, we are including Supplemental Video 1 of a juvenile female Sasquatch sleeping.  This video is from the Sasquatch that Sample 37 was obtained from which also lends credibility and chain of custody to that sample.  The still photos have been removed from the manuscript with the exception of Figure 4 which has now been edited to tie it to Supplementary Video 1 on lines 109-110 since the video is clearer and in hi def.

Below is the Journal Nature peer review with Dr. Ketchum's (authors') responses that I received from the anonymous source:

Author Responses to Referees 2

Referee #1 (Remarks to the Author):
Comments re: The sasquatch genome project
1.  Firstly - I reviewed a previous version of this ms and am pleased to see that the authors have wholly restructured their ms (it is now substantially improved, with many tangents and irrelevant areas of discussion removed or substantially modified). The previous version was rather opaque - was it dealing with sasquatch or not? I am pleased to see that this new version is bolder and more direct. However, does it present satisfactory data, and include satisfactory analysis of that data?

Given that much of the analysis and discussion concerns genetics, and given that I'm not a geneticist, my general feeling is that any decision about the fate of this ms must fall 'into the lap' of geneticist reviewers. Based on more general issues discussed here, I conclude that the work currently suffers from some areas of obfuscation that prevent me from understanding exactly what it is the authors are proposing. I therefore cannot recommend publication at the moment: if relevant geneticists argue for rejection of the ms, I would concur with this opinion. I admit to being greatly intrigued by the data being presented, however, and (as per last time) wonder if there is a 'Nature-worthy signal' in here somewhere.

The discussion early on of "high quality complete genomes" and so on sounds impressive, and I hoped to be impressed by the data collected and analysis of it. However, right out of the gate (1st line of abstract) we are told that the tissue samples concerned "were obtained... from... Sasquatch". This is problematic - the authors should state that the tissue samples were hypothesised to be from sasquatch, since the tissue samples were not collected directly from animals themselves: the project is testing the hypothesis that the samples come from an unidentified hominid.

Authors’ Response:
Though a number of the samples were taken immediately after eyewitness sightings of Sasquatch, we did change the verbiage to correct this, Line 36, “hypothesized to be” from Sasquatch.

2. The comment, also appearing early on in the ms (p. 4), that the DNA evidence reveals "human DNA interspersed with sequence [data] that is novel and distantly related to primates" raised my eyebrows - what do the authors mean by this? That sasquatch, if imagined to be verified as real, is a hybrid of human and non-primate ancestry? Non-primate? I was interested in seeing this qualified: does it stem from a misuse of the term 'primate'? (I think we all agree that humans are primates).

Authors’ Response:
Line 59: Corrected to read: human DNA interspersed with sequence that is novel but primate in origin.

3. It is further stated on p. 27 that phylogenetic results "indicate distant relationships with multiple primate sequences". Again - what exactly do the authors mean here? This is never explained and I regard it as a major problem - the universally favoured hypothesis (as made clear elsewhere in the present ms) is that sasquatch (if real) is a primate, and specifically a hominoid, and presumably a hominid, a hominine and perhaps a hominin, so the text creates the impression that a non-primate ancestry is being preferred. Or, again, is this because 'primate' is being used to mean 'non-human primate'?

Authors’ Response:
We omitted the word distantly in Line 584.  The novel primate lineage comes off in the non-human range even though there are human sequences interspersed in the genome. So, we were referring to non-human primate.


4.  I was hoping that this would be clarified by examination of the phylogenetic/gene trees. While I'm pleased that several phylogenetic/gene trees have been included, they desperately need redesigning, since the text on the branches is so small that it can't be read without exceptional magnification. As such, the trees are essentially useless. 

Authors’ Response:
We have attempted to enlarge them but in PDF, they can be enlarged in Reader.  We are still trying to make them easier to read and currently have them at a publisher attempting to enlarge them without changing the actual data and distance tree.

4. I appreciate that the authors have apparently tried to include as much data as possible in this ms. I would say that the photographic evidence (Fig 4A and 4B) should not have been included, since those images are poor in quality, do not appear compelling, and weaken the credibility of the ms. Scientific names are written incorrectly on p. 23. 

 Authors’ Response:
We removed the still of 4A and relabeled 4B as Figure 4.  Supplementary video 1 shows the same Sasquatch in greater detail and in high definition and therefore supports the use of Figure 4, however if requested, we will remove 4 also.  Since this individual is the donor of Sample 37, we felt that tying her to a photo and video was important.  We will remove Figure 4 if the editorial board or the reviewers believe that it should be removed after this explanation.  Lines 584 through 586 were corrected as far as the scientific names.

Referee #2 (Remarks to the Author):
Short list of some scientific problems:

1. Repeated and abusive use of vague terms such as "multiple", "numerous", "many" or strange scientific terms such as "anomalous", "strinking", "unexplained" or "unexpected".  

Authors’ Response:
The words “multiple”, “numerous” and “many” were either removed or greatly reduced in their usage.  "Anomalous", "striking", "unexplained" and "unexpected" were completely removed from the manuscript.   2. There is a complete lack of statistical evidence or referenced scientific work for the hair analysis; claiming "they were strikingly distinct from human hairs" without any reference or evidence beyond this bold, authoritative statement is not acceptable. 

Authors’ Response:
References 15-19 refer to the hair analysis. Figure 5 illustrates the differences between Sasquatch hair and human hair.  Materials and Methods also discuss the methods utilized for hair analysis.  Statistics are not utilized in forensic hair analysis.

3. The vagueness of the extraction procedures is also surprising (how the contamination was minimized or DNA recovery maximized?): "The DNA was extracted in a clean room using forensic science procedures that minimized contaminant DNA in the samples while maximizing DNA recovery."

Authors’ Response:
The Material and Methods were supplemental due to the length of the paper and it was stated in the manuscript.  The extraction techniques were discussed at length in the Supplemental Materials and Methods. 
 
4. Some paragraphs are confusing and again, the use of terms such as "clearly" or "as expected" is strange. "Control DNA was obtained from the majority of the submitters and was profiled using Promega PowerPlex16 20. As expected, all submitters yielded complete profiles. In contrast, when the hominin samples were tested using Promega PowerPlex16, partial profiles were evident in almost all cases. These data clearly showed that the samples were not contaminated by their submitters." (how?)

Authors’ Response:
The words clearly and as expected were removed.  Lines 265 and 266 were edited “These data showed that the samples were not contaminated by their submitters since all of the submitters’ profiles excluded as being a contributor to the hominin profiles/partial profiles.” to show how the samples were not contaminated by the submitters.

5. The authoritative and unsupported statements follow along the paper: "In the experience of this laboratory, hair samples constitute a very reliable source of DNA." 

Authors’ Response:
We had previously had the following in the paper but when we revised it, we discarded it due to the increased length of the manuscript, however, we have reentered it into the manuscript, Line 280-283: “Almost all large animal breed registries utilize plucked hair samples as their primary source of DNA for their parentage testing programs.  One horse registry alone processes over 100,000 hair samples per year.  These hair samples are archived and are viable for many years after they are submitted.  Therefore the hominin samples were well within our expectations as far as nuclear DNA yield and quality”. 

6. Some methodologies are totally unspecific and uninformative: "After extraction, yield gels with 3 L of the extracted DNA were utilized to determine if there was DNA present and whether it was degraded". 

Authors’ Response:
The Material and Methods discuss this and it is visualized in Figure 7.  Yield gels are a well established method for visualizing not only the quantity of DNA in an extraction but also the quality of the DNA since DNA will “smear” on a gel instead of giving a distinct band if the DNA is degraded.  Mild degradation is depicted in Figure 13 with the human control that had been purposely degraded prior to extraction.

7.  More vagueness (we don't know how many samples are all): "All of the screened samples revealed 100% human cytochrome b and hypervariable region 1 sequences". 

Authors’ Response:
Line 317 changed to “All 110 screened samples”.
8. More methodological problems; in degraded DNA, artifactual amplifications are expected and potentially undescribed alleles have to be sequenced to see what's in the PCR product. "PowerPlex16 amplification of the hominin samples yielded only partial profiles with off-ladder alleles while amplification of DNA."  ents : "Once it was established that the Sasquatch nuclear DNA did not conform to human DNA.."

Authors’ Response:
 The DNA was not degraded as we had a yield gel of the raw DNA showing clear bands (Figure 7) not smears. As some of us are forensic scientists, we are expert in determining DNA quality and mixture (contamination) interpretation and the interpretation of PP16 which we use in court for our DNA profiles.  We included references which support our statement as well as the peak heights on electropherograms showed adequate DNA where the amel X dropout should not have occurred (Figure 9).  We also sequenced amel X and the results are seen in Table 4. References 43-46 address the X  Dropout.  We can address this exponentially if necessary.

9. More vagueness: how were the complete mtDNA genomes obtained?  Apparently the samples were sent to a company, I guess they amplified it in overlapping fragments? of which length? Or were they captured with some enrichment method and posteriorly sequenced with next generation technologies? One cannot say the samples were sent and results sent back and that's it

Authors’ Response:
From Materials and Methods:  Aliquots of purified DNA from all of the samples in this study, along with human controls to monitor for possible contamination, were shipped to Family Tree DNA. Proprietary methods were used to amplify the mitochondrial DNA genome. The DNA was amplified using 48 sets of human specific primer pairs that overlapped. Extra primers were developed and utilized in case of failure due to mutation. The amplicons were sequenced on an Applied Biosystems® 3130xl Genetic Analyzer. 

10. Instead of generating meaningless mtDNA phylogenetic trees, the authors should check the well known mtDNA phylogeny (for instance, at PhyloTree) and tell us exactly the haplogroup, and/or subhaplogroup and haplotypes of each sample. It will be self evident anyway that these mtDNA genomes fell within the modern human variation, no need for any tree. 

Authors’ Response:
We were asked for the mtDNA phylogenetic trees in the first submission so we furnished them.  They were actually important since the same mtDNA sequences were found in the next generation whole genome sequencing as in the original mtDNA sequencing.  We furnished trees for both the original individual mtDNA sequencing as well as the sequence pulled from the whole genomes.  We furnished a table with haplogroups for the various samples (Table2).

11. Unsupported claims (by the way, allelic dropout is a well known phenomena in degraded samples and still the most plausible explanation): "It is noteworthy that AmelX allele dropout occurs in significant numbers of the unknown samples yet seldom occurs in normal human testing." 

Authors’ Response:
We have previously addressed that the samples were not degraded.  We also furnished references for the X dropout (References 43-46) and the peak height (RFU) of the Y peak precludes degraded or insufficient DNA as a cause of  X dropout (Figure 9).  The samples were tested twice with different methods (PP16 and amelogenin only) and the dropout repeated.  Amel X was also sequenced and it failed on most of the samples though the human controls provided normal sequence.  Three of the authors have seen literally thousands of PP16 profiles from both paternity and forensic applications.  Only one author has actually seen one case of X dropout and it was because of mutation, not degradation or low yield DNA.  From the paper: The genotyping of the amelogenin locus produced the most consistent results across the samples tested. The DNA samples yielded four types of results: XX, XY, Y and null. The dropout of the X amplicon was the most significant of the findings observed with the STR genotype analysis of Amelogenin. (Figure 9, Supplementary Data 3) This dropout was reproduced in several individual samples and was repeatable both in the multiplex of PowerPlex 16 and the analysis of the STR locus, so it is unlikely to be an experimental artifact due to low quantity or degraded DNA (Table 3). The repeatability and number of samples exhibiting the X dropout is inconsistent with what would be expected with normal human allele dropout43-46. It is noteworthy that AmelX allele dropout occurs in significant numbers of the unknown samples yet seldom occurs in normal human testing.

12. More vagueness (how long? how do you assess pristiness?): "DNA samples that yielded long and pristine sequences". 

Authors’ Response:
The quality of the DNA sequences can be assessed by viewing the electropherograms.  These are available if needed.  Also the Q30 score for the whole genomes denoted the extremely high quality of the sequences, Lines 544-558.

13.  More methodological problems (the observed -again, vague- results are likely due to the amplification of environmental and degraded DNA yielded unspecific PCR products):"The resulting sequences ranged from totally non homologous matches, not found in Genbankafter multiple BLASTs (including dissimilar sequence BLASTs) to novel SNPs and even failure to sequence" 

Authors’ Response:
The quality of the DNA was addressed using Figures 7 and 13 and the Q30 scores.  The novel sequences and SNPs repeated in the genomes although there was no failure in the whole genomes.  Failures in the early testing can be attributed to primer design failing to amplify novel sequence.

14. More methodological problems: the MC1R sequences (which primers?, which length?), come apparently from PCR products that were not cloned (this is an standard procedure while working on ancient DNA samples to ascertain the heterogeneities present in the PCR product and also the original extract). Thus the C to T change observed in two samples could be the result of cytosine deamination, a well known phenomenon in degraded DNA. In any case, what can be deduced from this section (and also from the mtDNA and the MYH16 section) is that this samples are modern human DNA samples. 

Authors’ Response:
We have previously discounted degradation of the DNA using visualization by yield gel and Q30 scores on the next generation sequencing.  Also, this is fresh, contemporary DNA, not ancient DNA.  Sample 26 was worked up thoroughly including histopathology that showed fresh tissue with no degradation or bacterial contamination.  Since a whole genome was sequenced with fresh tissue and still provided the same results as were obtained in the early testing, the only conclusion is that the genome is novel and high quality.  It is no different than any other genome sequenced today with fresh tissue or blood.  We have added the primers as Supplemental Data 9.

15. Meaningless experiments and sections (obviously the low performance in the SNP chip is due to low quality DNA): "In an effort to mimic severely degraded DNA that could explain the strikingly low SNP matches obtained, one of the human controls submitted along with the unknown hominin samples comprised non sterile blood that was purposely maintained at room temperature in a moist environment for 4 days in an effort to maximize degradation of the sample. Upon visual inspection, hemolysis of the sample had occurred and bacterial contamination, which often correlates with DNA degradation, was seen. An acrylamide gel was loaded with the degraded human sample to assess the degradation and was visualized with ethidium bromide. Smearing was observed".

Authors’ Response:
If the DNA is degraded, it will smear on a yield gel.   Yield gels have been used for years in forensics and standard DNA testing to assess DNA quality from fresh specimens. We have figures of two yield gels showing DNA that is not degraded and two samples that are showing some degradation, including the human control, Figures 7 and 13.

16. More meaningless sections (artifactual or missing bands are common in ancient DNA, this has been known for about three decades now): "Some of the samples appeared to produce normal amplicons that resulted in bands consistent with the human controls. Other samples displayed clear bands that appeared to be of different sizes than those expected of normal human amplicons. Yet others had multiple bands. Still other samples failed to amplify at all." 

Authors’ Response:
This is fresh DNA and fresh dried DNA and the degradation issue has already been addressed.   The same samples would sequence long pristine (via electropherogram) sequences up to 900 bases at other loci consistent with human as well as novel sequences hundreds of bases long. 

17. More vagueness (which ones?): "and forensic techniques to ensure that there was no human contamination"

Authors’ Response:
See Materials and methods: “Since the presence of normal human DNA contamination of submitted samples was a primary concern throughout this study, all samples were thoroughly cleaned in a manner consistent with forensic testing procedures. In order to further rule out contamination from human personnel and lab workers, samples from submitters and scientists working with the samples were collected for comparison with the results obtained in the various DNA tests.
Hair samples were then sorted into two groups for extraction at DNA Diagnostics. DNA from those samples containing 5-50 or more single hair roots were selected and the roots clipped into 1.5 mL microcentrifuge tubes. The hair roots were thoroughly cleansed with water and ethanol prior to extraction to remove any extraneous DNA.
Hair roots were placed in microcentrifuge tubes for DNA extraction and ATL buffer (Qiagen) was added. These samples were digested with proteinase K (PK, 20 mg/mL) and dithiothreitol (DTT, 1.0 M) at 56°C overnight, followed by a three-step organic extraction procedure using phenol:chloroform:isoamyl alcohol (25:24:1) with an additional PCI extraction. This process was followed by a butanol wash and buffer exchange/concentration into TE-4 buffer (10 mM Tris, 0.1mM EDTA, pH 8.0) using Microcon®-100 ultrafiltration devices (Millipore, Billerica, MA)92-93.
The remaining unknown hairs with only 1-5 hair roots were sent to the North Louisiana Criminalistics Laboratory (NLCL, Shreveport, Louisiana) for DNA extraction and purification. The roots were cleaned with water prior to digestion. The cleaned roots were digested in ATL buffer (Qiagen), PK, and DTT at 56°C until completely dissolved, which generally was overnight. The DNA in this crude extract was purified using the EZ1® DNA Investigator Kit with cRNA (Qiagen) and eluted into TE-4 on a BioRobot EZ1® (Qiagen).
Saliva swabs, blood swabs, and tissue cuttings (10 mg) were placed in microcentrifuge tubes for DNA extraction. The samples were extracted using the above mentioned organic method with the exception that DTT was not used during digestion.
Reference samples, in the form of buccal swabs from submitters who collected the unknown hair and tissue samples, were isolated using 50mM NaOH and heated to 100 for 10 minutes followed by the addition of 1M Tris (pH 8.3)98. The DNA extracted at DNA Diagnostics was quantified using a Nanodrop spectrophotometer (Thermo Scientific, Willimgton, DE). Hair samples sent to the NLCL were quantified by real time PCR using the Applied Biosystems Quantifiler® Human kit on an Applied Biosystems Prism® 7000 Sequence Detection System99.  Samples that yielded DNA concentrations too low to use in standard testing had their DNA concentration augmented using multiple displacement amplification method per the manufacturer’s instructions86.
DNA was then visualized on a 1% agarose gel to determine DNA quality by loading 3 ┬ÁL of DNA extraction.  Appearance of the bands determined the quality and quantity of the DNA extraction (Figures 7 and 9)”.
18. More vagueness: "According to the laboratory, the sequences themselves were of very high quality" 

Authors’ Response:
We have added information and the summary generated by the HiSeq 2000 Next Generation Sequencer showing that the genomes were of high quality via the Q30 score, Lines 478-492, and removed the statement above since it is no longer necessary due to the generated Q30 scores.

19. More methological problems (environmental, contaminating DNA will produce no matches at GenBank, this is common in all ancient DNA genomic projects): "Some sequences produced no homology matches when BLAST searched against all primate, human, Neanderthal, Denisova, and other sequences in Genbank."

Authors’ Response:
We previously addressed that 1. The DNA is not ancient and 2. The DNA was not degraded or contaminated both by visualization and testing in the case of preliminary findings and by the Q30 scores generated by the HiSeq2000 for the whole genomes.

20. Although they claim to have generated 30x genoms, I understand they have only assembled and analyzed the chromosome 11, they don't explain why. 

Authors’ Response:

We explained that chromosome 11 is highly conserved in primates.  In the paper: “Thus, the selective supercontigs comprised an abundance of neural associated and putative tumor suppressor sequences all of which are highly conserved in primates and humans and clearly establish that the Sasquatch is closely related to humans. The high homology with multiple primate lineages (including but not limited to, chimpanzee, macaques, gibbons and marmosets) and with humans as demonstrated in phylogenetic trees (Figures 19, 20, and 21) indicate that the supercontigs contain highly conserved human and primate gene sequences”.  
The goal of this manuscript was to prove that there exists an unknown primate living in North America since eyewitness reports consistently describe a creature with the appearance of a primate.  It takes years to analyze a genome so in order to prove that we had a novel primate, it stood to reason that an area of the genome that is highly conserved in primates be the first area of the genome to be investigated.  The supercontigs supported the existence of a novel primate using chromosome 11, which was the goal of this manuscript.  

21. The obvious explaination for this section is that the sample is a mixture of DNAs or has been contaminated at the Sequencing Service (did they use a tag sequence for this particular project?; was the service sequencing primates?): "the Sasquatch consensus sequence that showed homology to human chromosome 11 reference sequence is distantly related to multiple primate lineages including Homo Sapiens, Pan Troglodytes (Chimpanzee), Macaca Mulatta (Rhesus Monkey), Nomascus Leukogenys (White cheeked Gibbon) and Callithrix Jacchus (Common Marmoset)." 

Authors’ Response:
 The UT core lab only sequences human DNA and this statement has been added on Lines 472-473.  We also previously addressed that we have added the Q30 scores for the genomes.  These scores absolutely prove that there was not a mixture of species in the whole genomes as explained above. Lines 544-558 from the manuscript: The run summary generated by the HiSeq 2000 next generation sequencer provides scores, Q30, for run quality. Q30 can also be used to determine if there was any contamination (or mixture) found in the samples sequenced.  According to Illumina, a pure, single source sample would have an average Q30 score of 85. However, if there was contamination present in the sample sequenced, the divergent sequences would compete against one another causing a contaminated sample to have a Q30 score of 40 to 50.   The Q30 scores for the first read for the three genomes sequenced had Q30 scores of 92, 88 and 89 respectively.  The second read was slightly lower 88, 84.25 and 83.66, but still in line with the 85 average. The Q30 is the percent of the reads that have the statistical probability greater than 1:1000 of being correctly sequenced. Therefore, not only were the sequences from a single source, but the quality of the sequences were far above the average genome sequenced using the Illumina next generation sequencing platform. The high quality of the genomes can be attributed to the stringent extraction procedures utilized whereby the DNA was repeatedly purified.   This ultra-purified DNA also allowed for greater than 30X coverage of the three genomes.  The summary of the next generation sequencing generated by the HiSeq 2000 Illumina sequencer is furnished as Supplementary Data 7.

22. Sequencing data should be freely available to the scientific community after publishing (even better, before). 

Authors’ Response:
We attempted to upload all sequences to GenBank but they refused them (I have the email and it said because we do not have a species name).  We asked how to do it and they refused to return our emails or calls).  As a result, we attached all sequences as supplemental per Dr. Gee's request.

23. In my view, there conclusions are not supported by the data. What do we have here is likely low quality DNA samples belonging to modern humans that yield some conflicting results in unspecific genotyping approaches. I suggest also some background contamination in the next generation sequencing from previous primate sequencing projects, again likely influenced by the original low quality of the samples.

Authors’ Response:
This is an incorrect assumption as the data was repeatable and of good quality.  We have defended our position numerous times (above) and have added new data (Q30 scores) supporting our position that the DNA is not degraded nor is it contaminated.


Referee #3 (Remarks to the Author):

I appreciate the additional work the authors have undertaking trying to meet the concerns raised by me and the other reviewers. However, I am afraid that I do not find the paper worth publishing. The authors still lack explaining in any convincing way how this "new species" carries mtDNA genomes identical to those of modern humans only. I also believe that alternative explanations exist as to why the nuDNA genomes differs from that of contemporary humans e.g. mapping a mixture of animal DNA and human contamination to human reference genomes. 

Authors’ Response:
We felt that the data should speak for itself so we did not include a theory concerning the lack of novel SNPs in the mtDNA or the presence of the modern human mitochondrial DNA across all 110 samples.  We have added a theory to the Conclusions Section in the manuscript, Lines 734-738.

We also previously addressed that we have added the Q30 scores for the genomes.  These scores absolutely prove that there was not a mixture of species in the whole genomes as explained above. Lines 544-558 from the manuscript: The run summary generated by the HiSeq 2000 next generation sequencer provides scores, Q30, for run quality. Q30 can also be used to determine if there was any contamination (or mixture) found in the samples sequenced.  According to Illumina, a pure, single source sample would have an average Q30 score of 85. However, if there was contamination present in the sample sequenced, the divergent sequences would compete against one another causing a contaminated sample to have a Q30 score of 40 to 50.   The Q30 scores for the first read for the three genomes sequenced had Q30 scores of 92, 88 and 89 respectively.  The second read was slightly lower 88, 84.25 and 83.66, but still in line with the 85 average. The Q30 is the percent of the reads that have the statistical probability greater than 1:1000 of being correctly sequenced. Therefore, not only were the sequences from a single source, but the quality of the sequences were far above the average genome sequenced using the Illumina next generation sequencing platform. The high quality of the genomes can be attributed to the stringent extraction procedures utilized whereby the DNA was repeatedly purified.   This ultra-purified DNA also allowed for greater than 30X coverage of the three genomes.  The summary of the next generation sequencing generated by the HiSeq 2000 Illumina sequencer is furnished as Supplementary Data 7.

I want to thank the anonymous source for having the courage to come forward and give us vital information about the Ketchum DNA Study.  It is my desire that this information will help those in the Bigfoot Community and the general public at large to understand the study and the events that have occurred over the past nine months.









6 comments:

  1. Scott, please. You are obviously not familiar with the peer review process. "Accept with revisions" means it did NOT pass. It means the revision has to address the points by the reviewers so that it can be accepted. After correction, addition, the review starts all over again. The outcome could still be "Nope, not like this". Besides, this information was out there in December last year already.

    Source: I have published in journals myself.

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  2. I know exactly what this means Scott. Do not stray. The data in this review was only slightly questioned; alas, the grammar was actually requested to change more than. With the majority of Q30 scores contained in this ms, the data is not mostly erroneous, but rather correct. Most have no idea what CH11 is or what it does. Nor do they know what amel x allele is either. Carry on accordingly.

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  3. On the battlefield; stall, delay and harrass tactics
    serve to give advantage to a superior force that has not
    yet arrived. Superior or not the one wanted to win. This
    could happen in science, could it?

    ReplyDelete
  4. hopefully skyes will have simular findings as this study,
    at this point, with all the negative press, it's the only thing that is going to redeem it.

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  5. A bit confused here. Ketchum admitted on her site that she DID NOT pass peer review.

    http://www.advancedsciencefoundation.org/#!press-release/cm8a

    Are the above just made up lies? What's going on Scott?

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    Replies
    1. Ed, please stop lying and trying to confuse the reader, I checked this link of her so called "Journal". Dr. Ketchum did not set this up nor did she make an entry. The "admission" you are referring too is a statement made by "Melba's friends, family, and closest loved ones... "

      Below is the actual quote. If you cannot be more honest in the future I will have to block you from commenting. I do not mind you stating your opinion, but when you purposely attempt to mislead (LIE) then I will have to draw the line. Ed, you have been warned, do it again and your banned.

      Here is the quote:
      As Melba's friends, family, and closest loved ones... it became our intent to facilitate and establish an online platform where Melba could freely self-publish her ground breaking science. Her work and manuscript were rejected by NATURE in November of 2012, then rejected again by JAMEZ in January 2013, and promptly rejected again by another scientific online journal toward the end of January / beginning of February 2013. All of these three American Journals as well as the International Journals who rejected Melba should be boycotted for their blanted scientific bias against Melba and her work. She is a brilliant scientist, self-taught geneticist, and loving veterinarian.

      Delete