Tomorrow’s issue of the high impact & widly cited journal Cell hosts this paper, “A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing (DOI:10.1016/j.cell.2008.06.021)” First author, Richard Green, says that this genome is essentially without error. The genome comes from the Vindija 33.16 specimen, a 38,000 year old Neandertal from Croatia, of which around 0.3 grams of bone was extracted and mtDNA isolated.
Serre et. al. sequenced the HVR1 region of the mtDNA of the Vindija 33.16 sample in 2004, and Richard Green et al. sequenced 2414 bp of mtDNA sequence from this sample in the famous 2006 paper, “Analysis of one million base pairs of Neanderthal DNA.” Like the 2006 paper, 454 sequencing was used in the current paper because it doesn’t rely on cloning, and yet provides 34.9 fold coverage.
I won’t get into the nitty gritty details of the sequencing protocol, but here’s some of the conclusions of the mitochondrial genome analysis. Comparing the assembled 16,565 base pair Neandertal mtDNA sequence to the 16,568 base pair Cambridge reference mtDNA sequence (rCRS) showed that there are 206 differences, of which 195 are transitions and 11 are transversions).
To assess the evolutionary relationship between modern humans and this Neandertal, the authors compared this Neandertal mitochondrial genome to 53 different mtDNAs of extant humans as well as a bonobo and chimpanzee. They estimated the divergence time of the Neandertal mitochondrial genome by using the 6-8 million year old divergence time of chimpanzees. They estimate a 660,000 year old divergence time between humans and Neandertals, with a 95% credibility interval of 520,000–800,000 years ago.
The subunit 2 of cytochrome c oxidase (COX2), an enzyme that functions in the electron transport chain, of modern humans exhibits four amino acid substitutions compared to the Neandertals. While the authors don’t know the implications of these subsitutions, they do consider this interesting,
“…because mtDNA is inherited without recombination, and because the Neandertal mtDNA falls outside the variation of modern human mtDNA, this single modern human observation represents a reversion to the ancestral state seen in Neandertals and chimpanzees. Thus, these four amino acid substitutions occurred in the relatively short period after the divergence of Neandertal and extant human mtDNAs and before the most recent common ancestor of current human mtDNAs. The observation of four nonsynonymous substitutions on the modern human lineage, and no amino acid changes on the Neandertal lineage, stands in contrast to the overall trend of more nonsynonymous evolution in Neandertal protein-coding genes, and deserves consideration.”
How does Green know that the genome is without error? In other words, how do we know contamination ain’t an issue? 454 sequencing generates,
“a high average coverage of the random sequence reads in combination with ampliﬁcation and sequencing of positions where coverage is low, or where longer nucleotide homopolymers may cause base calling problems, make us conﬁdent that the error rates from both these sources are low.”
The authors estimate at most, modern DNA contamination is 0.5%.
I’d be really interested to see the sequences of Neandertals prior to the last Ice Age, when population sizes were relatively larger and the genetic diversity would be larger. DNA from Neandertals that were around 110,000 years ago would be great, because, what we have now, is from few individuals that were around glaciation would affect the observed mutations. Some of you may point me 2006 Current Biology paper by Orlando et al. where the authors were able retrieve 123 bp of the mtDNA HVR-1 from the molar of a 10-12 year-old Neandertal child from Scladina cave, Belgium… but that’s a very short region piece of DNA from an individual well inside the glaciation period… But I don’t know of any 110,000 year old Neandertal specimens off the top of my head.