RNA Data Show Ketchum Sample 26 - the Smeja Kill - is a Black Bear

ABSTRACT

The Ketchum Sample 26 nDNA sequence (2,726,786 bp long) was searched in total against the RNA reference genomic sequence (RNA ref_seq) database of the National Center for Biotechnology Information (NCBI), commonly known as GenBank.  The matches to polar bear were far better than any other species.  Primates, including human, were far poorer matches than the next best non bear matches.   A phylotree showed Sample 26 in precisely the position that a black bear would occupy.  Since Sample 26 is from California and since there are no black bear data in this database, the polar bear is the surrogate match from the same genus.  Sample 26 is a black bear. 

INTRODUCTION

The conclusion of the Ketchum paper, “Novel North American Hominins…..” [1], unsupported by any published sequence comparisons, was that:

“…the species (sasquatch) possesses a novel mosaic pattern of nuclear DNA comprising novel sequences that are related to primates interspersed with sequences that are closely homologous to humans.” 

However, we showed previously, with multiple sequence comparisons, that Sample 26 (S26) – the Smeja kill – is a bear, most likely a black bear (Ursus americanus)[2], in agreement with three separate laboratory analyses [3], and that Sample 140 is a dog [2]. Only Sample 31 matched a human best [2]. Primates, including humans, were not even close to matching Samples 26 and 140. Reference [2] involved milking the “Nucleotide”, “Transcriptome shotgun analysis” (TSA) and “Ref_seq genomic” (RSG) databases. At that time all polar bear data was confined to the TSA database and was missed by the Ketchum team, and there was little black bear data in any of these databases. Consequently our best hits were polar bear (Ursus maritimus) or giant panda (Ailuropoda melanoleuca). We updated our results later when a whole polar bear genome was added to the RSG database [4]. As expected it was now the best hit, as it is in the same genus (Ursus) as the black bear, whereas giant panda is not.

Later still, we searched the “Expressed sequence tags” (EST) database, which has black bear data, and found it matched S26 better than any other species.[5] For sequences where there was no black bear data, the best other species hits were outmatched by polar bear data from the RSG database. In only one out of 59 best hit sequences was a dog the best match. Other results showed the presence of human, dog, and bear DNA in S26 [6] and the fallacy of the Ketchum consultants’ methodology [7]. Other problems with the mtDNA were also addressed [8], including degradation [9].

The volume and consistency of these results should have convinced anyone of the bear origin of S26. However, some remain unconvinced. See Reference [10] for Melba Ketchum’s reasons for remaining so. Hence, we search yet another database here, the “RNA ref_seq) database. The database contains RNA sequences, including polar bear but not black bear. RNA (ribonucleic acid) is the complement of DNA (deoxyribonucleic acid): bases U for A or A for T and C for G or G for C). It uses three-base codons for amino acids to manufacture proteins, the workhorses of the cell. Any biochemistry text will explain this in more detail. The take away point here is that these RNA sequences can be searched against DNA queries – the software makes the base conversions mentioned above. In this paper we search the previously untapped RNA ref_seq database with the entire 2,726,786 bp S26 nDNA sequence from [1]. Our results and conclusions corroborate our previous findings, but contrast sharply with the Ketchum et al. conclusion above.

COMPUTATIONAL METHODS

 

All searches were conducted with the BLAST™ software (http://blast.be-md.ncbi.nlm.nih.gov/Blast.cgi) of the NCBI, and hits were downloaded as Excel files as described previously [2].   Search parameters were default except that for whole S26 sequence searches maximum hits was set to 5000 and word size to 64.  This is the same search software used by Ketchum et al. [1].  Her claim that “He didn't use bioinformatics software which has to be done in order to evaluate the data.”[10] is false.  The “BI” in NCBI means “biotechnology information” = "bioinformatics."  One does not need extra “bioinformatics” software to compare two numbers such as %IDs or scores. The hard part, which Ketchum et al. totally failed at, is generating relevant sequences to compare through appropriate searches.  The phylotrees (Figs. 1and 2) were constructed just as the Ketchum Supp. Figs. 5 and 6 (reproduced here as Figs. 3 and 4) were with the BLAST™ feature “Distance tree of results”, as discussed below.

Another Ketchum claim, “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.” is a red herring. My procedures as described in [1], and used throughout, do not involve “tearing it down into little sequences”. That was her consultants’ approach, which was decscribed by an NCBI contact of ours as “makes no sense”. Only in [7] did we break the Ketchum nDNA sequences down to compare results to her consultants’. Even then I got better matches to a bear and a dog for S26 and S140, respectively, with their methodology. You just have to search in the right databases. Incidentally, statistics are widely used in genetics, including the use of the Poisson distribution of mutations, which is what we did in [8] to show that some of the Ketchum mtDNA sequences were outside the range of normal humans. They were not our “only” tool, and in fact were “only” used once in the case of the mtDNA sequences in [8].

Our computational methods are sound and used by geneticists everywhere in some form or another. We applied them across multiple databases and made conclusions based on known phylogeny and taxonomy. Ketchum et al. did not.
 

RESULTS AND DISCUSSION

The RNA ref_seq database was queried with the entire S26 sequence, 5000 max hit entries, word size = 64, limited to mammals. A list of 27,135 total hits (more than one per database entry) was downloaded and sorted by score, then %ID. The best 30 hits by score were culled and examined. Table 1 shows the best 15 hits (plain text). Ten were polar bear (the only bear in this database), three had no polar bear sequences in the same range, and two had shorter, but higher %ID polar bear matches (in bold italics). Over these latter five ranges searches of the Reference genomic sequences (refseq_genomic) database limited to bears produced the underlined hits in Table 1. In these cases, polar bear was the best match by score and %ID.

The RNA ref_seq database was again queried limited to polar bear. The best 15 hits are listed in Table 2 (plain text). Ten of these are the same as the polar bear hits in Table 1. The best non polar bear hits over the same hit ranges from the mammals list were added to Table 2 (bold italic). A query limited to human only was performed and the best human matches over the same hit ranges were added to Table 2 and underlined. Ten of these sequence ranges matched ranges for best hits in [1] or [5]. In every case the polar bear was the best match by score and %ID. Human matches were a distant third.

Phylotrees are comparisons to multiple species, based on pairwise comparisons to their many sequences, and as such offer much more proof of identity than any single match does. A phylotree (distance tree of results) was produced from BLAST™ results (mammal) as Fig. 1. It clearly shows S26 in a close phylogenetic relationship to carnivores, especially bears. Notice the very distant relationship to all primates, including human. See the NCBI taxonomy database for comparison [11]. The unopened leaves (not shown in Fig. 1) revealed many of the species seen previously based on the RSG database [7] and in the same relative positions. Fig. 2 shows the expanded carnivore leaves from Fig. 1. Notice, as expected, walrus, seal, dog, ferret, polar bear and panda from Table 2. Contrast these phylotrees with the Ketchum et al. conclusion in the INTRODUCTION, and with the meaningless Ketchum phylotrees (Supplemental Figures 5 and 6 in [1]) as discussed in [7]. Those phylotrees are reproduced here for comparison as Figs. 3 and 4, taken from [7] and based on [1]. As we commented before,[7] “Simply stated, chicken, fish, mouse, and human are too distantly related to each other to be, as a group, the most related species to the Ketchum Sample 26.” “Where’s everything else?”

For comparison to Fig. 2, Fig. 5 shows the currently accepted Ursus phylogeny compared to a few select other carnivores [12]. The correspondence of S26 in Fig. 2 to black bear in Fig. 5 is excellent and unequivocal. Other species are also in the same relative positions in both figures.

CONCLUSIONS

The polar bear is the best RNA ref_seq match to S26. There are no black bear data in this database. However, the phylotree – distance tree of results – shows S26 in exactly the position that would be occupied by a black bear, distinct from the polar bear. Also, because the sample was collected in California, we believe it is a black bear, the only extant bear there. This was the fifth database in GenBank which supports a bear conclusion unequivocally. Also, three independent laboratory DNA analyses indicated a black bear [3].

Again we see the need to do multiple structured searches, sometimes against multiple databases, and to download and sort the hits to unravel the identity of a sequence, especially in the light of conserved genes. Settling for a single search of a single database such as our “mammals only” search and only looking at the top score in Table 1 and not %ID would have led to a false conclusion that the Pacific walrus (Odobenus rosmarus divergens) was the best match. This was the mistake of Ketchum et al. when they only searched the Nucleotide database (which contained no polar bear data and little black bear data) and concluded a human match for this sample even though the %IDs averaged only 94-95%, as explained previously [2, 7]. A species match requires 99+%ID. As noted previously, “One cannot match what is not in the database.” [2]

The Ketchum et al. conclusion, “…the species (sasquatch) possesses a novel mosaic pattern of nuclear DNA comprising novel sequences that are related to primates interspersed with sequences that are closely homologous to humans.” IS WRONG. Sample 26 is from a black bear. 

ACKNOWLEDGEMENT

The author received no financial or other material support for this work.

CONFLICT OF INTEREST

The author declares no conflicting interests

REFERENCES


[1] See Sasquatch Genome Project link at right.

[2] See Paper 1 links at right.

[3] See The “Tyler Huggins Report” under Pages at right and on this blog, November 26, 2014, “Ketchum Sample 26, The Smeja Kill: Independent Lab Reports.”

[4] See on this blog, November 30, 2014, “Table 1 Updated: The Ketchum Sample 26 nDNA.”

[5] See on this blog, May 22, 2015, “New Black Bear Data Show Ketchum Sample 26 (the Smeja Kill) is a Bear.”

[6] See Paper 3 link at right.

[7] See on this blog, December 30, 2014, “Melba Ketchum’s Experts and Their Mistakes: What’s in a Phylotree.” 

[8] See Paper 2 link at right.

[9] See on this blog, December 29, 2014, “Melba Ketchum Shows Sample 140 Degradation in her YouTube Video.”

[10] See on this blog, September 1, 2014, “My Response to Melba Ketchum’s Facebook Post About Me.” 

[11] For taxonomy see: http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=33554.

[12] Cronin, M. A. et al., “Molecular Phylogeny and SNP Variation of Polar Bears (Ursus maritimus), Brown Bears (U. arctos), and Black Bears (U. americanus) Derived from Genome Sequences, Journal of Heredity 2014:105(3), pp. 312–323.


Table 1.  Best 15 Mammal Hits

Accession [a]         %ID[b] LEN[c] MIS[d] GAP[e]  Start[f]     End[g]   Score[h]   Species

XM_004394587.1 no match, RNA NW_007929448.1	96.58 98.83	2136 2134	45 0	2 1	189026 189028	191136 191136	3515 3779	Pacific walrus polar bear polar bear
XM_008704386.1	97.86	1679	2	4	1655920	1657569	2870	polar bear
XM_008688345.1	98.9	1460	1	2	1761456	1762900	2593	polar bear
XM_008686832.1	98.6	1359	1	1	759948	761288	2388	polar bear
XM_008701063.1	99.05	1267	2	2	855387	856643	2265	polar bear
XM_004752061.1 no match, RNA NW_007907318.1	95.82 99.56	1388 1374	39 3	9 2	312149 312149	313520 313520	2224 2501	domestic ferret polar bear polar bear
XM_011235586.1	99.65	1149	4	0	2258573	2259721	2100	polar bear
XM_008688389.1	99.23	1174	4	1	1835440	1836608	2113	polar bear
XM_003780852.1	94.43	1364	48	9	1657662	1659004	2073	galago
XM_008704386.1	99.67	304	1	0	1657662	1657965	556	polar bear
NW_007907230.1	99.87	787	1	0	1658089	1658875	1448	polar bear*
NW_007907230.1	99.77	429	1	0	1657662	1658090	787	polar bear*
XM_008707444.1	98.96	1151	0	2	1508093	1509231	2049	polar bear
XM_008686832.1	99.64	1104	3	1	756646	757748	2015	polar bear
XM_004412889.1	97.3	1183	23	2	602055	603228	1999	Pacific walrus
XM_008686943.1	99.89	888	1	0	602341	603228	1635	polar bear**
NW_003218343.1	98.99	1183	3	2	602055	603228	2109	giant panda+
XM_008690191.1	99.81	1050	2	0	2257336	2258385	1929	polar bear
XM_008688318.1	100	1040	0	0	2586084	2587123	1921	polar bear
XM_011750120.1 no match, RNA NW_007907318.1	97.83 100	1108 1103	20 0	1 0	363058       363059	364161     364161	1910 2037	macaque polar bear polar bear

 Footnotes [a] - [h]: Same as in Table 2. 

 *  Combine
**  Shorter sequence, but better %ID match.
+   Polar bear match had long gap, probably a sequencing error.
galago is short-eared galago.
macaque is pig-tailed macaque

Table 2.  Best 15 Polar Bear Hits

Accession [a]      %ID[b] LEN [c] MIS[d] GAP[e]   Start[f]    End [g]  Score[h]    Species

XM_008704386.1	97.86	1679	2	4	1655920[i]	1657569	2870	polar bear
XM_011222467.1	96.45	1688	7	13	1655920	1657569	2736	giant panda
XM_011545195.1	92.27	1695	73	30	1655921	1657569	2351	human
XM_008688345.1	98.9	1460	1	2	1761456[i]	1762900	2593	polar bear
XM_002920733.1	97.67	1460	19	2	1761456	1762900	2494	giant panda
XM_011542584.1	92.46	1459	95	2	1761457	1762900	2071	human
XM_008686832.1	98.6	1359	1	1	759948[i]	761288	2388	polar bear
XM_004755948.1	98.08	1353	11	8	759948	761288	2340	domestic ferret
XM_011519835.1	94.76	1354	35	8	759948	761288	2074	human
XM_008701063.1	99.05	1267	2	2	855387[i]	856643	2265	polar bear
XM_006728292.1	97.71	1266	18	3	855387	856643	2167	Weddell seal
XM_011520245.1	95.03	1268	48	8	855386	856643	1978	human
XM_008688389.1	99.23	1174	4	1	1835440[i]	1836608	2113	polar bear
XM_002925708.2	98.64	1174	11	1	1835440	1836608	2074	giant panda
NM_001261833.1	93.87	1175	65	3	1835440	1836608	1764	human
XM_008690191.1	99.65	1149	4	0	2258573[i]	2259721	2100	polar bear
XM_004412631.1	98.96	1149	12	0	2258573	2259721	2056	Pacific walrus
XM_011542831.1	96.34	1147	42	0	2258575	2259721	1886	human
XM_008707444.1	98.96	1151	0	2	1508093[i]	1509231	2049	polar bear
XM_004795929.1	96.04	1161	16	11	1508093	1509231	1862	domestic ferret
XM_005274051.2	93.29	1147	62	6	1508092	1509231	1677	human
XM_008686832.1	99.64	1104	3	1	756646[i]	757748	2015	polar bear
XM_004406102.1	98.82	1104	12	1	756646	757748	1965	Pacific walrus
XM_005252729.2	96.74	1104	35	1	756646	757748	1838	human
XM_008690191.1	99.81	1050	2	0	2257336	2258385	1929	polar bear
XM_011235586.1	99.43	1050	6	0	2257336	2258385	1906	giant panda
XM_011542831.1	95.24	1050	50	0	2257336	2258385	1663	human
XM_008688318.1	100	1040	0	0	2586084	2587123	1921	polar bear
XM_002912684.2	99.42	1040	6	0	2586084	2587123	1888	giant panda
XM_006718826.2	96.06	1040	41	0	2586084	2587123	1694	human
XM_008704414.1	98.67	1054	0	3	1663527[j]	1664579	1857	polar bear
XM_002917304.2	97.91	1054	8	3	1663527	1664579	1812	giant panda

XM_005274336.2	94.53	1060	49	8	1663527	1664579	1628	human
XM_008690205.1	99.31	1012	4	1	2102443[j]	2103451	1827	polar bear
XM_011233674.1	98.62	1015	8	3	2102443	2103451	1792	giant panda
NM_001301044.1	94.22	1021	44	10	2102443	2103451	1544	human
XM_008711943.1	99.58	954	3	1	184464[j]	185417	1738	polar bear
XM_005633808.1	99.27	958	5	2	184460	185417	1729	dog
NM_001172705.1	98.12	956	15	3	184463	185417	1663	human
XM_008686832.1	98.95	953	1	4	761289	762232	1696	polar bear
XM_002917485.2	98.53	954	4	5	761289	762232	1676	giant panda
XM_011519835.1	95.61	957	22	14	761289	762232	1517	human
XM_008701063.1	97.41	1005	5	2	856642	857646	1694	polar bear
XM_004790381.1	97.2	1000	28	0	856642	857641	1692	domestic ferret
XM_011520245.1	94.8	1000	52	0	856642	857641	1559	human

[a] In NCBI Genbank..
[b] Percentage of matching base pairs (bp) over the sequence range.
[c] Sequence length, bp.
[d] Mismatches, bp.
[e] Number of gaps (not number of bp in gaps)
[f] Starting position of match in bp along S26 sequence.
[g] End position of match in bp along S26 sequence.
[h] Score, see NCBI BLAST™ Handbook.
[i] Same sequence range in Paper 1.
[j] Same sequence range in EST blog, May 23, 2015.

Fig. 1.  Phylotree of All Mammal Hits

Fig. 1.  Taken directly from BLAST(TM) Distance tree of results with common names added.   Distance to European rabbit on top was shortened to compact.  "Unknown" = S26.



Fig. 2.  Expanded Phylotree - Carnivores Only

Fig. 2.  Taken directly from BLAST(TM) Distance tree of results with common names added in red.


Fig. 3.  Ketchum Supp. Fig. 5 as Redrawn in [7]



Fig. 3.  Ketchum Supp. Fig. 5 [1] as redrawn in [7].  Unknown sample number was not stated in the Ketchum paper.  Numbers  are distances (fraction of mismatches) to the unknown, calculated from the original Ketchum Supp. Fig. 5.          
 



Fig. 4.  Ketchum Supp. Fig. 6 as Redrawn in [7]




Fig. 4.  Ketchum Supp. Fig. 6 in [1] as redrawn in [7].  Unknown sample number was not stated in the Ketchum paper.  Numbers  are distances (fraction of mismatches) to the unknown, calculated from the original Ketchum Supp. Fig. 6.          


Fig. 5.  Select Carnivore Phylotree [12]

Fig. 5.  Taken unchanged from Ref. [12]. Not a complete phylotree of all known species.  Other carnivores were omitted, e.g. pinapeds (seals, walrus) and more bears.   Domestic ferret (Fig. 2) is a subspecies of Polecat (Fig. 3).  ABC = Admiralty, Baranof, and Chichagof Islands (in southeast Alaska) where brown bears have significant polar bear DNA due to past hybridization.