Neanderthals and Cro-Magnon

Hi Quetzal,The contamination problems (plural as there are many different problems) not only applies to old or ancient DNA but to modern samples as well. However, the low copy and degraded state of ancient DNA exacerbates the problem. Iwill try below to show why human or primate ancient DNA is particularly tricky relative to say work with mammoths.Problems:1. There is more DNA in a few cells of skin you shed than there probably is in most ancient human fossils. What this means is by the time you get the fossil from somebody who will allow you to whack it to pieces to extract DNA from it, lots of people have touched it with bare hands..not to mention those who did the excavation. If you cannot sample from an internal part of the sample i.e. taking a bone core then you will have a major contamination source. As Cooper in the Nature commentary states and has been demonstrated by others over the years, soaking in acid, bleach, UV light does not necessarily get rid of all contamination.2. PCR primers that work for humans usually work for their relatives or near relatives. If I design PCR primers for mtDNA of Homo sapiens..they will work for Cro-Magnon as well. Contrast this to mammoths. I can design (and have frequently) primers that are specific for elephants. You can spike a reaction with human DNA and still never amplify it..so my Russian curator friends can touch the bones all they want and it won't cause me any problems.3. Mungo Lake suffered from a different problem which plagues ancient DNA as well which are nuclear insertions of mtDNA or NUMTS. These are copies of mtDNA that have wandered into the nucleus and are now pseudogenes...there are hundreds if not thousands in the human genome...they often look very much like human mtDNA for example but often show differences from most bona fide mtDNA...they can be so divergent that they fall outside of the genetic variation of mtDNA for the human species i.e. like the neandertal type specimen sequence. That is why Krings et al. spent so much time and experimentation to support that the neanderal sequences was not an insert. The Mungo Lake sequence phylogenetically clustered with a known human mtDNA NUMT which is a big heads up that you have contamination...the most famous example is the claim that dinosaur DNA had been sequenced and it turned out to be a human nuclear insert sequence..doh!With the above in mind...what do you do with ancient human samples? If the samples is contaminated your negative controls could still look good since your reagents are presumably not contaminated. You extract from a Cro-Magnon or a neandertal and get a sequence that looks like an average Joe mtDNA sequence. You send it to another lab to replicate the result (and since they will have the same contaminated sample) they may reproduce you contamination result...you can test multiple different samples and see if the results are consistent i.e. is the next Cro-Magnon sequence from another location similar or radically different? But if it comes back as a different common human mtDNA haplotype..what then? If it is completely different...which sequences is correct? Is one a contaminant? Are both?Thus, you end up only accepting sequences that look very different from modern human mtDNA and that can be confirmed as non-nuclear in origin...and I think it is pretty pointless to do that much work if you cannot accept basically an entire array of data as valid because of contamination fears. Of course accepted neandertal sequences will be different from modern human since if a neandertal sequence with modern human sequences is found it will be dimissed as contamination!Contamination, cross contamination, NUMTS all plague modern DNA studies as well...but a modern sample usually has so much DNA in it that you would really have to contaminate it pretty badly. Numts are a bigger problem which the scientific community mostly ignores rather than addresses...for my part, I have switched to nuclear DNA markers for mammoth analysis since my work with elephants showed they are so loaded with NUMTS that it is almost impossible to figure out what the bona fide mtDNA sequence is for the regions of the mitochondria that would interest me...of course, since so few mammoths yield nuclear DNA it has slowed my research down..but I rather deal with that then publish a completely screwed data set...and besides..its not like there is a big community of people working on mammoths...so my competition tends to be minimal compared to my more medically oriented work..so I can afford to be more plodding in my progress ...how is that for an excuse for being lazy

Perhaps, however, the objectors are memmbers of the one group that has the most to lose which is the group that sequenced the first neandertal and want it to be different. However, the objectivity in the process comes from the dozens of labs that have attempted to pick apart the neandertal results..and thus far have been unable to..in fact, every subsequenct neandertal sequenced has reinforced the conclusions of the first study. The point will soon be moot as using a new pyro sequencing method that was used to sequences about 1% of the mammoth genome, the neandertal nuclear genome is being queued up for sequencing. If they get enough sequence, this will provide the kind of data necessary to determine if the "different species" status neandertals currently enjoy based on mtDNA is supported by nuclear markers. Or perhaps nuclear genes will support neas as a different species but that there was some admixture. Remains to be seen.

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It seems to me then that drawing conclusions from DNA studies of Neanderthals is a severely flawed process.

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Going in to the DNA based projects, nobody had any idea what the results would be..the results have been scritinized by dozens of groups and has held up...more groups are scritinizing the results still. The Cro-Magnon results have not been held to this kind of scritiny yet.Science is not like religion where when something is wrong in religion, it is covered up. There is always somebody out there in science trying to knock down your conclusions or expand on your findings...or beat you to it. Not the mindless orthodoxy demanded by conservative relgions.There is nothing flawed about the conclusions drawn. From the 7 or so neandertals sequenced from very different localities thus far, there is absolutely no evidence of either being human or having mixed with human populations. However, the conclusions are tentative for the reason I gave that it is not possible to distinguish a contaminant from endogenous DNA if the sequence were to be more human like...thus far this has not occurred for neandertals but is exactly what happened with Cro-Magnon. Whole genome (or large scale)sequencing will resolve the issue....again, science moves forward...you should try it sometime.Edited by Mammuthus, : No reason given.

As I mentioned in my response to randman, the nuclear genome of the neandertal type specimen is being cued up for sequencing. This will provide the kind of information that is needed to address the question of admixture. What you describe is seen in the two species of African elephants, the forest and savana elephants. It is called cytonuclear disocciation. What happens is that the much larger savana bulls outcompete the smaller forest bulls in areas where the two species meet. The resulting hybrids are half forest half savana at nuclear loci but pure forest for mtDNA. What has happened as a result is that you have some savana elephants that have almost completely savana nuclear genomes (because of backcrossing of to savana males) but have forest mtDNA.It may turn out that something like this exists between humans and neandertals i.e. there may be a few human individuals with nea like mtDNA but pure human nuclear and vice versa...or it could be that there was zero admixture.

Hi Jor-El
Regularly reported and verified extraction and characterization of DNA from samples in the 40-50,000 year old range are abundant i.e. woolly mammoths. I think about the oldest reproduced sequences came from 80 Ky bears. The limit will probably be around 100 K years unless a find is made in an unusual preserving context. Note, the one such context examined, insects encased in amber, did not pan out as none of the studies were reproducible.Most ancient DNA studies focus on samples in the age range from early Holocene to late Pleistocene (anywhere from a few thousand years to about 30,000 years ago).

I doubt there will even be Pleistocene or early Holocene Parks made. Even if one were able to sequence the entire genome of say a mammoth, how would you actually make a mammoth? So far cloning has involved nuclear transfer...not just blasting DNA into a denucleated cell...maybe someday down the road..but not anytime soon.Jurassic DNA probably completely degraded in the Jurassic regardless of how well preserved (morphologically) insects in amber may be.