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Friday, June 17, 2016

More Inconsistencies and Evidence for Contamination in Ketchum et al. Supplementary Figures

ABSTRACT

Scientific results should be consistent, otherwise more experiments are needed to clarify discrepancies.  The Ketchum et al.(2013) Supplementary Figures 1, 2, and 3, which are mitochondrial DNA phylotrees for samples 26, 31, and 140, respectively, are inconsistent with Supplementary Figures 7, 8, and 9, respectively.  Haplogroups of the closest relatives do not agree: Supp. Fig. 1 with 7 (S26), 2 with 8 (S31), or 3 with 9 (S140). The Most likely explanation is contamination by at least one human in each case.

INTRODUCTION 

Ketchum et al. (2013) mitochondrial DNA results have been previously reviewed (Paper 2 at right), and the Ketchum claim that they are all 100% human was found to be an overstatement.  In fact, it was found that eight of 18 samples with complete mitochondrial sequences had too many mutations from the closest haplogroup to be statistically probable (less than 1% probability).  Also, eight of 11 samples with HVR-1 (hypervariable region 1) only mutations listed were phylogenetically ambiguous, i.e., alternate haplogroups were equally likely.  From these new results, it was concluded that either the samples were contaminated and/or degraded, or that any possible hybridization events would have to have been followed by subsequent mutations along nonhuman evolutionary lines and on a different time scale.  The results in the current paper prove that S31 has human contamination by an individual with a different haplogroup than previously reported for that sample.  Samples 26 and 140 have previously been shown to be from a black bear and a dog, respectively, from nuclear DNA matches (See Paper 1 at right).  They are now shown here to be contaminated by two humans of different haplogroups.

METHODS

This study involves extracting data from six circular phylotrees, Supp. Figs. 1, 2, 3, 7, 8, and 9 from Ketchum et al. (2013) for comparisons.  Phylotrees of this kind are generated from the query results (hits) in BLAST (TM) through the "Distance tree of results" option.  The goal was to determine the haplogroup [1] of the nearest match to each query: S26, S31, or S140 in Supp. Figs. 1 and 7, 2 and 8, and 3 and 9, respectively.  Phylotrees in Supp. Figs. 1, 2, and 3 were generated from complete mitochondrial sequences produced by Family Tree DNA.  Phylotrees in Supp. Figs. 7, 8, and 9 [3] were generated from supercontigs, but the details of which mitochondrial genes were employed were not stated.

The title of the nearest phylotree branch tip was searched in GenBank (R)(the NCBI databases), and the accession number retrieved.  A BLAST(TM)[3] alignment of this accession with rCRS (Revised Cambridge Reference Sequence, Genbank accession NC_012920.1) produced a set of rCRS-based mutations, as seen in the "Graphics" option of the results page.  From these mutations a haplogroup was determined using the programs FASTmtDNA and mtDNAable as previously described (Paper 2 at right).        

RESULTS


Sample 26

Table 1 presents the results for S26. The nearest haplogroup from Supp. Fig. 1 (H1)closely matches that determined by Family Tree DNA (H1a).  However, The Supp. Fig. 7 result, T2b, is far removed.  Interestingly, this is the haplogroup of the human contamination determined by two independent studies (Cassidy, 2013; Khan and White, 2012-the Tyler Huggins Report at right).  Also to be noted is that S26 is one of the samples previously found to have too many extra mutations (16) to be called "modern human," according to the accepted mtDNA phylotree (van Oven, 2010) and a Poisson Distribution of mutations (Paper 2 at right).  Given that the nuDNA of this sample matched a black bear (Paper 1 at right; Cassidy, 2013;  Khan and White, 2012; Sykes et al., 2014 - See Tyler Huggins Report and Sykes Paper at right), it can be concluded that there are two sources of human contamination in this sample, with haplogroups H1a/H5e and T2b.

Table 1.  S26  
Nearest Matches to S26 mtDNA in Ketchum phylotrees
Supp.
Accession
Mis.vs.
Hap.
Fig.
S26
 
 
 
 
 
Homo sapiens clone 3760 mitochondrion,
1
JQ703795.1
16
H1
complete genome
Homo sapiens isolate NEC20 mitochondrion
7
JQ664540.1
22
T2b
complete genome
                  From Ketchum Supp. Data 2:
H1a
S26 from mtDNAable:
H5e


 

 
Columns left to right: Accession title, Ketchum et al. Supplementary Figure number,
Accession number (GenBank), Mismatches vs.S26, Haplogroup.
 
 


Sample 31

Table 2 presents the results for S31.  The nearest haplogroup from Supp. Fig. 2 (L1a1) is close to that determined by Family Tree DNA (L0d2a). However, The Supp. Fig. 8 results, T2b and T2b8, are far removed. The nuDNA of this sample matches modern human (Paper 1 at right).  Sample 31 is contaminated by another human of T2b haplogroup.




Table 2.  S31

 

 

 

 

Nearest Matches to S31 mtDNA in Ketchum phylotrees

Supp..

Accession

Mis. vs.

Hap.

Fig.

S31

 

 

 

 

 

Homo sapiens haplotype A10L1A2 mitochondrion

2

AY195777.1

2

L0d2a1

complete genome

(A10L1A2)*

Homo sapiens isolate 157 T2i Tor354 mitochondrion

8

JQ798131.1

100

T2 or T2b-16362C

complete genome

(T2i)*

Homo sapiens isolate 13T mitochondrion

8

 

 

complete genome

JX081995.1

104

T2b8

 

 

 

 

 

 

 

From Ketchum Supp. Data 2:

L0d2a

S31 from mtDNAable:

L0d2a1

*(Haplogroup) taken from Accession




Sample 140

Table 3 presents the results for S140.  The nearest haplogroup from Supp. Fig. 3 (D4b2b1) matches that determined by Family Tree DNA for HVR-1 only(D). However, The Supp. Fig. 9 results, both R2'JT, are far removed. The nuDNA of this sample matches a dog (First paper at right).  This sample is contaminated by two humans with haplogroups D and R2.





Table 3.  S140

Nearest Matches to S140 mtDNA in phylotrees

Supp.

Accession

Mis vs

Hap.

Fig.

S140

Homo sapiens mitochondrial DNA complete genome

3

AP008361.1

No complete sequence available**

D4b2b1

isolate PDsq0023

Homo sapiens isolate R1 mitochondrion,

9

JX155264.1

R2'JT(R2a1)*

complete genome

Homo sapiens isolate R2 mitochondrion

9

JX155265.1

R2'JT(R2a1)*

complete genome

           From Ketchum Supp. Data 2:

D (HVR-1)

      From Behar, et al.(2012):

D (HVR-1)

 

 

 

 

 

*  (Haplogroup) taken from Accession

**  Oddly Supp. Figs, 3 and 9 require a full sequence, but Supp. Data 2 contains only HVR-1 mutations


CONCLUSION


Over all three samples, using supercontigs resulted in phylotrees with haplogroups which were inconsistent with full sequence derived haplogroups. 

Samples 26 and 140 are contaminated by two modern humans.  Sample 31 is contaminated by one additional human.

Insistence by Dr. Melba Ketchum, DVM, that her samples were not contaminated when analyzed is not warranted.  Very likely some additional Ketchum et al. anomalous mtDNA samples are so because of contamination (Paper 2 at right).

NOTES

[1]  Haplogroups are unique human mtDNA sequences, represented by their mutations from a standard, either rCRS (revised Cambridge Reference Sequence) or RSRS (Reconstructed Sapiens Reference Sequence).  All known haplogroups of modern humans are represented in the phylotree of van Oven (2010) at www.phylotree.org.  This tree stems from the root called "Mitochondrial Eve", the most recent common maternal ancestor (MRCA) of all humans.  A haplotype is a particular allele (combination of SNPs-mutations) within a haplogroup and is designated by a preceding letter and number.

[2]  Supp. Figs. 7, 8, and 9 are erroneously referred to in the Ketchum et al. (2013) text as Supp. Figs. 4, 5, and 6 in the last paragraph of the "Next Generation Whole Genome Sequencing" section.  Supp. Figs. 4, 5, 6 are actually nuDNA-based phylotrees. See my blog "Melba Ketchum's Experts and Their Mistakes: What's in a Phylotree."    

[3]  BLAST (TM) is a search/match program which utilizes the National Center for Biotechnology Information (NCBI) GenBank databases. (Altschul et al.,1990; Madden, 2003).  Its application has been described extensively on this blogsite. See under BLAST Search and Ketchum DNA Study Tabs above.

REFERENCES 



  Altschul, S. F.; Gish, W.; Webb, M.; Meyers, E. W.; Lipman, D. J. (1990). Basic local alignment search tool.  Journal of Molecular Biology, 215 (no.3): 403-410.

  Behar D.M.; van Oven, M.; Rosset, S.; Metspalu, M.; Loogväli, E.-L.; Silva, N. M.; Kivisild, T.; Torroni, A.; Villems, R. (2012)  A “Copernican" reassessment of the human mitochondrial dna tree from its root.  American Journal of Human Genetics, 90 (no.4): 675-684. http://dx.doi.org/10.1016/j.ajhg.2012.03.002

  Cassidy, B. G. (2013).  Technical Examination Report DNAS Case Number: 2012-006524.  DNA Solutions, Inc. (Oklahoma City).

  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/view-dna-study/
 

  Khan, T. and White, B.  (2012)  Final report on the analysis of samples submitted by Tyler Huggins. Wildlife Forensic DNA Laboratory Case File 12-019, Trent University Oshawa (Peterborough, Ontario, Canada). http://www.bigfootbuzz.net/bart-cutino-tyler-huggins-release-sierra-kills-sample-dna-results/

  Madden, T.  (2003). The BLAST sequence analysis tool.  The NCBI Handbook; McEntyre, J; Ostell, J., Eds.; National Center for Biotechnology Information (Bethesda, MD). http://www.ncbi.nlm.nih.gov/books/NBK21097/.

  Sykes, B. C.; Rhettman A.; Mullis, R. A.;  Hagenmuller, C.; Melton, T. W.; Sartori, M. (2014) Genetic analysis of hair samples attributed to yeti, bigfoot and other anomalous primates.  Proceedings of the Royal Society B, 281: 20140161.


  van Oven, M. (2010).  Revision of the mtDNA tree and corresponding haplogroup nomenclature. Proceedings of the National Academy of  Sciences USA, 107 (no. 11): E38-E39.   http://dx.doi.org/10.1073/pnas.0915120107