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Author Topic:   Comparisons of Neandertal mtDNA with modern humans and modern chimpanzees
Loudmouth
Inactive Member


Message 31 of 80 (105041)
05-03-2004 6:24 PM


Is the data reliable?
I was perusing and came across this abstract, released on the web March this year:
Mol Biol Evol. 2004 May;21(5):957-64. Epub 2004 Mar 10.
Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences.
Pusch CM, Bachmann L.
Institute of Anthropology and Human Genetics, Division of Molecular Genetics, University of Tubingen, Tubingen, Germany.
Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood. [emphasis mine]
I was wondering if someone was familiar with this study. It would seem to me that exogenous DNA contamination could be a very large problem, and how previous studies may have overcome this problem. This would also seem to cast doubt on the repeatibility of the sequence data between the three known samples of neanderthal mitDNA.

Replies to this message:
 Message 36 by Mammuthus, posted 05-04-2004 3:57 AM Loudmouth has replied

  
redwolf
Member (Idle past 5791 days)
Posts: 185
From: alexandria va usa
Joined: 04-13-2004


Message 32 of 80 (105045)
05-03-2004 6:38 PM
Reply to: Message 27 by AdminSylas
05-03-2004 5:22 PM


Re: Off topic. posting new proposals
The reason I ask, is that I thought I'd posted a request for a thread on the subject of evolution and paranormal phenomena yesterday, and I can't find any trace of it. Either I missed something in trying to post it or it fell into some sort of bitbucket. Could you possible see if you see that anywhere?

This message is a reply to:
 Message 27 by AdminSylas, posted 05-03-2004 5:22 PM AdminSylas has replied

Replies to this message:
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AdminSylas
Inactive Member


Message 33 of 80 (105052)
05-03-2004 7:01 PM
Reply to: Message 32 by redwolf
05-03-2004 6:38 PM


Re: Off topic. posting new proposals
It was approved, and has already generated responses. It appears at Message 1
I've checked, and this new thread does not show in your list of posts. That's bad. I'll be asking Percy to check it out.

This message is a reply to:
 Message 32 by redwolf, posted 05-03-2004 6:38 PM redwolf has replied

Replies to this message:
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redwolf
Member (Idle past 5791 days)
Posts: 185
From: alexandria va usa
Joined: 04-13-2004


Message 34 of 80 (105058)
05-03-2004 7:32 PM
Reply to: Message 33 by AdminSylas
05-03-2004 7:01 PM


Re: Off topic. posting new proposals
>I've checked, and this new thread does not show in your list of posts...
That's another sort of a question I've had. Most forums have some sort of way to see recent posts and replies for the given user and I don't see that here, and that could be because a lot of what I see on evcforum is in terribly small fonts, at least on firebird. Do you have such a feature?

This message is a reply to:
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Replies to this message:
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jar
Member (Idle past 394 days)
Posts: 34026
From: Texas!!
Joined: 04-20-2004


Message 35 of 80 (105059)
05-03-2004 7:43 PM
Reply to: Message 34 by redwolf
05-03-2004 7:32 PM


Re: Off topic. posting new proposals
Well, I use Firefox and it all seems okay.
To see recent posts, just click on the users name and it will show the most recent in up to 30 treads.
For example, clicking on redwolf shows...
Posts by redwolf
edited to correct Firebird to Firefox.
{Added by AdminSylas: Can people please take discussion of this to followup from Message 27? Thanks. Even on Firefox, I think that the list will (at the time of posting) omit a link to redwolf's thread on "Evolution and paranormal things". jar -- if you can confirm seeing redwolf's post on "Evolution and paranormal things" in your list, please let me know in a followup to the message I have linked above. Thanks -- AdminSylas}
[This message has been edited AdminSylas, 05-03-2004]

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Mammuthus
Member (Idle past 6475 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 36 of 80 (105140)
05-04-2004 3:57 AM
Reply to: Message 31 by Loudmouth
05-03-2004 6:24 PM


Re: Is the data reliable? Yes and No.
It is worth noting that ALL ancient DNA sequences should be considered tentative. The paper you have cited has already generated a response
Mol Biol Evol. 2004 Apr 14 [Epub ahead of print] Related Articles, Links
Mutations Induced by Ancient DNA Extracts? Letter.
Serre D, Hofreiter M, Paabo S.
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Germany.
We have investigated whether some factor in ancient DNA extracts induces site-specific mutations in modern DNA as described by Pusch and Bachmann (2004). We find no evidence for higher mutation rates when extracts from three different Pleistocene mammals are added to modern DNA than when water or extraction blanks are added. We also fail to find evidence that any such factor affects ancient DNA sequences determined from the same extracts. This as well as the patterns of nucleotide substitutions seen in DNA sequences determined from hundreds of other specimens leads us to doubt that a previously unknown mutagenic factor can be a general feature of extracts from old tissues.
Thus, the results of the study you cite were in themselves, non-reproducible. Such "spiking" of DNA with ancient extracts does produce some artifacts like "jumping PCR"
Paabo S, Irwin DM, Wilson AC.
DNA damage promotes jumping between templates during enzymatic amplification.
J Biol Chem. 1990 Mar 15;265(8):4718-21.
But nothing like what Pusch and Bachmann report.
However, there are three (at least)major problems with ancient DNA even disregarding the study of Pusch and Bachmann
1. Nuclear copies of mtDNA (Numts) which can be extracted from ancient materials
Greenwood AD, Capelli C, Possnert G, Paabo S. Nuclear DNA sequences from late Pleistocene megafauna.
Mol Biol Evol. 1999 Nov;16(11):1466-73.
(check out the woolly mammoth Numt sequence in the sequence figure)
or which can totally flub up your phylogeny with modern DNA when comparing your sequences to ancient sequences
Greenwood AD, Castresana J, Feldmaier-Fuchs G, Paabo S. A molecular phylogeny of two extinct sloths.
Mol Phylogenet Evol. 2001 Jan;18(1):94-103.
(check out the anteater Numt figure)
2)contamination with modern DNA
Zischler H, Hoss M, Handt O, von Haeseler A, van der Kuyl AC, Goudsmit J. Links
Detecting dinosaur DNA.
Science. 1995 May 26;268(5214):1192-3; author reply 1194.
(the most famous screw up in ancient DNA...other than the work on amber)
3) non-random deamination, mutation during PCR amplification from ancient DNA extracts
Again see the pattern of substitutions in the sequence figures of
Greenwood AD, Capelli C, Possnert G, Paabo S. Nuclear DNA sequences from late Pleistocene megafauna.
Mol Biol Evol. 1999 Nov;16(11):1466-73.
And here is a study that quantitates the problem
Hofreiter M, Jaenicke V, Serre D, Haeseler Av A, Paabo S. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.
Nucleic Acids Res. 2001 Dec 1;29(23):4793-9.

This message is a reply to:
 Message 31 by Loudmouth, posted 05-03-2004 6:24 PM Loudmouth has replied

Replies to this message:
 Message 37 by Sylas, posted 05-04-2004 5:17 AM Mammuthus has replied
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Sylas
Member (Idle past 5260 days)
Posts: 766
From: Newcastle, Australia
Joined: 11-17-2002


Message 37 of 80 (105143)
05-04-2004 5:17 AM
Reply to: Message 36 by Mammuthus
05-04-2004 3:57 AM


Re: Is the data reliable? Yes and No.
(Added in edit... fixed the source to be "Sylas")
Mammuthus writes:
It is worth noting that ALL ancient DNA sequences should be considered tentative. ...
This is a very good point, and your post gives some well known cautionary tales.
There has been an attempt to test for some of the problems you mention. For example
Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans
by D. Caramelli et. al., in PNAS, May 27, 2003, vol. 100, no. 11, pp 6593-6597
Extract from the paper:

Authentication Methods: An Overview. Genetic typing of ancient samples is technically challenging, because DNA is generally degraded and present in small amounts in the available specimens. The most stringent standards for authentication of ancient DNA (23, 24, 25) were therefore followed in this study. In particular, using the same order and names of the nine key criteria described in ref. 23, we proceeded as follows.
  1. DNA was extracted in a laboratory room exclusively dedicated to ancient DNA analysis (Physically isolated work area).
  2. For each sample, two independent DNA extractions were performed from fragments of different bones, and PCR controls produced negative results (Control amplifications).
  3. Amplification of large DNA fragments, unusual in ancient DNA analyses, was not observed, and the final consensus sequences make phylogenetic sense, i.e., do not appear to be a combination of different sequences, resulting from contamination of the specimens by exogenous DNA (Appropriate molecular behavior).
  4. All results were identical in two independent extractions and two independent amplifications using four different overlapping primer pairs (Reproducibility).
  5. Ninety-three and 72 clones were analyzed for Paglicci-25 and Paglicci-12, respectively; the average rate of Taq misincorporation across fragments was low (4.3 substitutions every 1,000 bp within the HVRI), with at least 79% of the clones showing the consensus nucleotide at each DNA fragment (Cloning).
  6. A single DNA extraction and amplification of two overlapping fragments was independently repeated in a different laboratory for Paglicci-25; the sequences were consistent across laboratories (Independent replication).
  7. The degree of racemization for three amino acids was low in both samples, suggesting a high probability to obtain intact ancient biomolecules from the specimens (Biochemical preservation).
  8. The estimated copy number of target DNA (between 1,000 and 1,500 in both samples) was larger than the threshold under which sporadic contamination cannot be excluded (Quantitation).
  9. No human sequence was amplified from the horse remains found associated to the Paglicci skeletons using either primers specific for humans or for horse; on the other hand, the DNA sequence obtained from the horse remains using specific primers aligns well with sequences of Equus caballus in GenBank (Associated remains).
The nine key criteria agree in indicating that the authenticity of the sequences we present is supported as much as technically possible at the present. We are not aware of any other published study of ancient DNA considering all of the precautions we used here to exclude contamination.
This paper, by the way, is a comparison of Neandertals with contemporaneous modern humans (Cro Magnon). The conclusions are summarized in figure 2 of the paper. The following image is a link to the source, which is given in a new window. The associated description is quoted from the paper.
Average genetic distance vs Age"Average genetic distance between ancient and modern samples (2,566 sequences of modern Europeans; y axis), as a function of the samples' age (x axis, in thousands of years). Vertical lines represent two standard deviations above and below the mean. Squares, anatomically modern humans. Diamonds, Neandertals. The Paglicci samples typed in this study are indicated by open squares. The point at 0 years indicates the average pairwise difference between present-day samples."
The diagram shows a sudden discontinuity for Neandertals, and a consistency for anatomically modern humans which indicates that the methods for handling ancient DNA are probably reliable.
Cheers -- Sylas
This message has been edited by Sylas, 05-04-2004 09:53 PM

This message is a reply to:
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Mammuthus
Member (Idle past 6475 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 38 of 80 (105149)
05-04-2004 7:17 AM
Reply to: Message 37 by Sylas
05-04-2004 5:17 AM


Re: Is the data reliable? Yes and No.
Another criteria (though not one that is universally applied) is that an independent laboratory reproduces the result i.e. the chance that two labs get mislead by the same contamination is vanishingly small for example
Note here, a lab in Rome and a lab at the AMNH in New York both retrieved the same sequences from 3 mammoths in Figure 2.
Greenwood AD, Lee F, Capelli C, DeSalle R, Tikhonov A, Marx PA, MacPhee RD.
Evolution of endogenous retrovirus-like elements of the woolly mammoth (Mammuthus primigenius) and its relatives.
Mol Biol Evol. 2001 May;18(5):840-7.
This was also the case with the original nea paper from Krings et al.,
a part of the sequence was reproduced in the lab of M. Stoneking in Penn State.
Though a cumbersome extra step, it goes a long way to confirming the sequences obtained.
I should also note, that Krings et al. to their credit, were able to rule out (to a fairly high level of certainty) that the neandertal sequence was a Numt as they tested neandertal specific primers on a large panel of human DNAs of different ethnic origin and were never able to produce a product.
Unfortunately, this type of rigor was not applied to the Mungo lake specimen which generated a sequence that is almost identical to a known human Numt and thus I (and others) are sceptical as to its ancient origins.
Adcock GJ, Dennis ES, Easteal S, Huttley GA, Jermiin LS, Peacock WJ, Thorne A. Mitochondrial DNA sequences in ancient Australians: Implications for modern human origins.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):537-42. Erratum in: Proc Natl Acad Sci U S A 2002 Jan 8;99(1):541.
In using ancient DNA to study human origins, one has an additional problem. Any sequenced retrieved is expected to be at least reasonably similar to modern human sequences. Thus, contamination may resemble the "expected" sequence and makes it harder to determine the authenticity of the sequence obtained. Issues of DNA damage and Numts make things monsterously complicated. This is less of an issue with other fauna. If you get a human-like sequence from a mammoth it means you screwed up and you know it right away. Now, if you could just get dolphins to do ancient human DNA work you would be in the clear

This message is a reply to:
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Loudmouth
Inactive Member


Message 39 of 80 (105215)
05-04-2004 12:25 PM
Reply to: Message 36 by Mammuthus
05-04-2004 3:57 AM


Re: Is the data reliable? Yes and No.
Mammuthus and Sylas,
Thank you both very much. I had a strong suspicion that possible contamination or chemical alterations to the ancient DNA was well understood, but I wasn't sure on the specifics. The paper I cited in my post sounded pretty fishy, and it seemed this sort of problem would be easy to control for (especially if modern human DNA is the suspected contaminant). Thanks again, and I bow before your collective expertise , and I will definitely have to check out Mam's refs when I have a chance.

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Loudmouth
Inactive Member


Message 40 of 80 (105216)
05-04-2004 12:29 PM
Reply to: Message 37 by Sylas
05-04-2004 5:17 AM


Re: Is the data reliable? Yes and No.
quote:
This paper, by the way, is a comparison of Neandertals with contemporaneous modern humans (Cro Magnon).
Bingo, this ends the debate in my opinion, along with the other controls you listed. If there were a problem with ancient DNA (non-random PCR mutations) it should have reared it's ugly head in the Cro Magnon PCR's as well.
PS. See mssg 39 as well.

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Mammuthus
Member (Idle past 6475 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 41 of 80 (105217)
05-04-2004 12:31 PM
Reply to: Message 37 by Sylas
05-04-2004 5:17 AM


Re: Is the data reliable? Yes and No.
quote:
The diagram shows a sudden discontinuity for Neandertals, and a consistency for anatomically modern humans which indicates that the methods for handling ancient DNA are probably reliable.
I should have addressed this earlier so as not to generate so many independent posts and offend AdMoose by actually saying anything.
This issue of reliability is not the biggest weak spot of neandertal and Cro-Magnon studies. Soon after the Cro-Magnon study came out, Alan Cooper and Svante Pbo wrote an op-ed objecting to the study on the grounds that if a sequence from Cro-Magnon is retrieved that is similar to modern human DNA sequences, it cannot be distinguished absolutely from a potential contamination and therefore must be rejected as authentic. Unfortunately (for them) this could be equally applied to the neandertals that Pbo studied. If you get a sequence from a neandertal specimen under rigorous lab protocol that is right within the middle of modern human genetic variation, it will be dismissed as a contaminant by many if not all the aDNA community (and won't be published). Why does this matter? If you can only accept sequences that are blatantly different from the human mtDNA gene pool as authentic and the question you are asking is "did neandertals contribute to the human mtDNA gene pool?" then your only possibility is to answer no. This is not scientific if you can throw out any data that points to a "yes" answer as was done with the Cro-Magnon results.

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Mammuthus
Member (Idle past 6475 days)
Posts: 3085
From: Munich, Germany
Joined: 08-09-2002


Message 42 of 80 (105219)
05-04-2004 12:34 PM
Reply to: Message 40 by Loudmouth
05-04-2004 12:29 PM


Re: Is the data reliable? Yes and No.
It still leaves the question open as to what exactly was in the extracts that Pusch and Bachmann were using? One that mutates naked DNA non-randomly? In my own experience I have seen random mutations among clones and in some cases a prevalence of C-T transitions (as is expected chemically anyway), but extracts that generate specific misleading haplotypes?
[This message has been edited Mammuthus, 05-04-2004]

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Loudmouth
Inactive Member


Message 43 of 80 (105224)
05-04-2004 12:53 PM
Reply to: Message 41 by Mammuthus
05-04-2004 12:31 PM


Re: Is the data reliable? Yes and No.
quote:
Soon after the Cro-Magnon study came out, Alan Cooper and Svante Pbo wrote an op-ed objecting to the study on the grounds that if a sequence from Cro-Magnon is retrieved that is similar to modern human DNA sequences, it cannot be distinguished absolutely from a potential contamination and therefore must be rejected as authentic.
Good point. You would think that there would be detectable differences between Cro-Magnon sequence and modern human sequence, such as y-chromosomal mutations as well as mitDNA mutations. Or even better, recent retrotransposon activity:
Ovchinnikov I, Troxel AB, Swergold GD. Genomic characterization of recent human LINE-1 insertions: evidence supporting random insertion. Genome Res. 2001 Dec;11(12):2050-8.
However, reliably dating recent mutations and insertions could be just as unreliable as the aDNA itself, so maybe we are stuck with the very problem that you mention.
One of the controls that Sylas mentioned was large DNA fragments (I am guessing > 5kb). This would seem to be a reliable indication of modern contamination, no matter the DNA source. I would assume that aDNA is around 100 to 500 bp, and anything over 5kb would automatically raise eyebrows (much less intact chromosomal DNA at 25 to 30 kb).
I think that the objections raised by Cooper and Paabo are important, but it seems that it can be worked around.

This message is a reply to:
 Message 41 by Mammuthus, posted 05-04-2004 12:31 PM Mammuthus has replied

Replies to this message:
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 Message 47 by Mammuthus, posted 05-05-2004 12:05 PM Loudmouth has replied

  
jar
Member (Idle past 394 days)
Posts: 34026
From: Texas!!
Joined: 04-20-2004


Message 44 of 80 (105252)
05-04-2004 2:31 PM
Reply to: Message 43 by Loudmouth
05-04-2004 12:53 PM


Re: Is the data reliable? Yes and No.
Would it give more information if DNA samples from contemporary samples were used.
What I was wondering was just how much difference there is between a DNA sample from a 40,000 year old Homo Sapiens and one from today?

Aslan is not a Tame Lion

This message is a reply to:
 Message 43 by Loudmouth, posted 05-04-2004 12:53 PM Loudmouth has replied

Replies to this message:
 Message 45 by Loudmouth, posted 05-04-2004 3:00 PM jar has replied

  
Loudmouth
Inactive Member


Message 45 of 80 (105258)
05-04-2004 3:00 PM
Reply to: Message 44 by jar
05-04-2004 2:31 PM


Re: Is the data reliable? Yes and No.
quote:
What I was wondering was just how much difference there is between a DNA sample from a 40,000 year old Homo Sapiens and one from today?
Figure 2 from the paper Sylas references in his post, I get the following data (approximations from the graph, not raw data).
Sample--------------Age-------Difference from modern humans
"old" Homo________25,000 yo___________0.010
Neandertal________29,000 yo___________0.085
Difference = average pairwise difference per base (I think).
And there is no statistical overlap between the two groups (old homo and neandertal. Given that the samples are separated by 4,000 years I would say you could count this as contemporaneous, or at least close to it. If there were random interbreeding between Cro-Magnon (old homos) and Neandertals then these numbers should be a lot closer. Also, the differences amongst modern humans appears to be the same as differences between modern humans and Cro-Magnon. The figure in Sylas's post has a box on the y-axis. That is the current difference among humans, and it is level with the box at 25 (25,000 years old on the x axis). I am not sure if there were anatomically modern humans 40,000 years ago, but I think the study cited here might help clarify things a bit.
Added in edit: Sylas's post = Message 37 in this thread. He has a hyperlink to the full paper. Figure 2 from that paper is what I am referencing.
[This message has been edited Loudmouth, 05-04-2004]
{AdminSylas makes a link. By writing [msg=-37] you give a link to a message in your thread. See ubbcode for full description.}
This message has been edited by AdminSylas, 05-05-2004 12:54 AM

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