Location of Ust’-Ishim Femoral shaft discovery 7
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 Geographic location.
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 Bone Morphology. Lateral View Posterior View Cross-section
Sample extraction 10 Two samples at different time points: 890 milligrams (mg) & 450 mg Collagen preservation satisfied criteria established by Oxford Radiocarbon Accelerator Unit (ORAU) Acid/Base wash, then gelatinize sample Ultrafiltration Freeze-dry AMS (Accelerator Mass Spectrometry) measure 14 C levels
14 C dating Age of Ust’-Ishim was estimated at 41,000 ± 1,300 years for 1 st extract 41,400 ± 1,400 years for 2 nd extract Fluctuation in 14 C levels throughout time Eg. Late 1800s use of coal diluted biosphere levels of 14 C Data must be calibrated to known carbon levels at that time Use of tree ring data to establish atmospheric carbon levels Marine fossil records 13
Oxcal-Calibrated results 14 Ust’-Ishim calibrated to 45,000 years BP Likelihood distributions Variance between actual and calibrated is width of blue line
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 Dating North greenland ice core project (NGRIP)—measure of stable 18 O to obtain climate information up to ~120K years (others up to 800K years) NGRIP data
DNA retrieval & sequencing 9 samples from distal part of femur were removed and libraries were constructed. Adaptors were ligated to the DNA and double indexed 16
Library construction & verification of human DNA Illumina HiSeq 2000 ~1.8-10% could be mapped to human genome 8 more libraries constructed from sample with the highest proportion of human DNA These treated with Uracil-DNA glycosylase and endonuclease VIII Removal of deaminated cytosine as a result of damage <35 mers in library depleted by acrylamide gel electrophoresis 17
Sequencing Results 42-fold sequence coverage of ~1.8GB of autosomal genome Coverage of X and Y chromosomes was ½ indicating bone was from a male <1% of the DNA fragments were a result of contamination 18
Population relationships Ust’-Ishim is more similar to present day Eurasians than Africans 7.7 positions/10,000 are heterzygous in Ust’-Ishim Ust’-Ishim mtDNA belongs to the R haplogroup (Western Eurasia) 9 directly carbon dated ancient mtDNA estimates age of bone to be consistent with radiocarbon date Thus, two confirmations of age 1 AMS mtDNA 19
Principal Component analysis Measure the variance between Ust’-Ishim and other populations Genome and genotype data for 922 present-day individuals from 53 populations 20
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 PC analysis Ust’-Ishim more closely related to non-Africans
How is Ust’-Ishim genome related to present human genomes? 87 African and 108 non-African individuals D-statistics to analyze derived alleles with present day humans Confirms that Ust’-Ishim is more closely related to non- Africans Ust’Ishim is equally or closely related to present day Asians and 8K to 24K western Eurasians but not present day Europeans Supports the hypothesis that part of European Ancestry did not participate in the initial dispersal from Asia and Europe. 22
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 Statistics testing whether the Ust’-Ishim genome shares more derived alleles with one or the other of two modern human genomes (X, Y).
Mutation Rates High quality genome sequence and carbon dating methods : Find the number of missing mutations between present day humans and Ust’-Ishim 0.44 X 10 -9 to 0.63 X 10 -9 per site per year Influenced by error rates Pairwise Sequentially Markovian Coalescent (PMSC) Measures coalescence time between 2 chromosomes to estimate past population changes Less error prone 24
Neaderthal admixture Admixture of Neanderthal and modern humans ~37K-86K BP Could Ust’Ishim predate this admixture? It does not as the percentage of derived alleles between Neanderthal and Ust-Ishim is 2.3% Ust-Ishim lived closer to time of admixture, thus Neanderthal DNA will be less fragmented than present day genomes 26
Q Fu et al. Nature 514, 445-449 (2014) doi:10.1038/nature13810 Regions of Neanderthal ancestry on chromosome 12 in the Ust’-Ishim individual and fifteen present-day non-Africans.
Summary of Results Carbon Dating and mtDNA analysis confirms Ust’-Ishim femur to be ~45000 BP More closely related to Eurasians Ust’-Ishim belonged to a population at same time or close in time of divergence between West and East Eurasians Determination of mutation rate that is consistent across all non-African genomes Ust’-Ishim does not predate Neanderthal admixture 29
Carbon Dating continued Ratio can determine age Biosphere has fluctuated : example use of coal diluted the 14 carbon pool in the late 1800s Cities have different ranges Nuclear war How is this accounted for? Best way is to have a carbon record for the atmoshpere frpom the time period—use of tree rings and fossils (good to 13,900 years) other fossils (marine) have been used to date back to 50K. (although not atmospheric there is a correlation delay between marine and atmoshperic that can estimate atmospheric levels Anyway-build a calibration curve that more accurately describes our data. Oxcal software makes these graphs: 30