Anthropology.net

Last month I was excited to share some research about the chemical composition of Ötzi, the 5,000 year old Tyrolean Iceman that has captured my attention for quite sometime. Today, I’m even more excited to share that the complete mitochondrial genome of Ötzi has been sequenced using a combination of PCR amplification and 454 sequencing. The research has been published in Current Biology. You can find it under the title, “Complete Mitochondrial Genome Sequence of the Tyrolean Iceman.”

I’ve covered the details behind Ötzi before. I’ll give you a quick run down in case you forgot or never knew about him. Ötzi is the name given to mummy discovered on September 19^th, 1991, around 3,270m above sea level, in the Eastern Alps near the Austro-Italian border. His remains were dated to be , 5,350–5,100 years old, and was remarkably preserved because of the cold climate. We have an idea what his last meal was and what he wore. He’s thought to have been around 46 years old, his fertility has been questioned, and his cause of death seems to have been rather horrific — severely wounded by an arrow and some blunt force trauma to his face.

Previous researchers have sequenced some of his mitochondrial genome, specifically the hypervariable segment (HVS-I). Two nucleotide transitions, at positions 16224 and 16311, indicate that Ötzi’s mtDNA belonged to haplogroup K, a subclade of the major west Eurasian haplogroup U. The authors of the new Current Biology paper decided to completely sequence the mitochondrial genome of Ötzi using 454 pyrosequencing technology. They’ve compared the sequence to 115 published complete mtDNA sequences from modern individuals, and constructed a phylogeny of the K haplogroup.

The sequencing run seems to have been rather uneventful. I’ve covered 454 technology before, but to recap it is sequencing by synthesis, which involves template DNA being immobilized, and solutions of each nucleotide added. They hybridize to their complement at the first unpaired base of the template. The hybridization reaction lets off light, because the polymerase enzyme is paired with another other chemiluminescent enzyme. A high resolution photo is taken and any remaining unbound nucleotide is removed and then another wash of another base is made. With the array approach developed by 454, it is possible to generate over 100 million nucleotide data in a 7 hour run with a single machine.

The authors reported they made 45,829 reads, but only 42,695 reads, or 93.2% of the total, were usable. Several gaps in the mitochondrial genome were observed, so PCR products were cloned into vectors and sequenced with conventional Sanger technology. When compared with the revised Cambridge Reference Sequence (rCRS), the consensus sequence showed 30 mtDNA transitions. A phylogenetic comparison was made to all 115 haplogroup K complete sequences currently available. The authors confirm that Iceman’s sequence falls within haplogroup K.

But, transitions at positions 3513 and 8137 on the Iceman’s mitochondrial genome indicate that his maternal lineage belongs a K1 subhaplogroup but not to any of the three subclades into which K1 is currently further subdivided (K1a, K1b, and K1c). The authors conclude that the Iceman’s mtDNA, belong to a novel branch of K1, not yet identified before. They’re calling it K1ö.

I’m not gonna get into much of a discussion about contamination because the samples were taken from thawed tissue from the mummy’s rectum. Many lines of evidence show that there was a lot of endogenous mtDNA that woulda muddled out any contaminating DNA and the results reconfirm the previous HVS-1 results. Anyways, this is the oldest complete H. sapiens mtDNA genome generated to date. The results show that as the frequency of genetic lineages change over time, due to genetic drift, some variants die out. Based upon the mtDNA, it is highly unlikely that Ötzi has any modern day maternal relatives… unless we sequence more than 115 haplogroup K carriers.

Luca Ermini, Cristina Olivieri, Ermanno Rizzi, Giorgio Corti, Raoul Bonnal, Pedro Soares, Stefania Luciani, Isolina Marota, Gianluca De Bellis, Martin B. Richards, Franco Rollo (2008). “Complete Mitochondrial Genome Sequence of the Tyrolean Iceman” Current Biology, DOI: 10.1016/j.cub.2008.09.028