1. Neanderthals Noonan, et al. Sequencing and Analysis of Neanderthal Genomic DNA Green, et al. Analysis of one million base pairs of Neanderthal DNA Kristine Briedis Journal Club 11/21/06

2. Neanderthals Most closely related hominid group to modern humans First recognized 150 years ago in Germany Existed 500,000 years ago Lived in Europe and West Asia Evolved away from humans Disappeared 30,000 years ago http://www.msnbc.msn.com/id/13154583/

3. Interbreeding? Open question if humans and Neanderthals cohabitated Overlap in their geographical ranges and time of existence Some late Neanderthal groups adopted cultural traits that might have been influenced by modern humans

4. DNA Before now, Neanderthal DNA studies were limited to mitochondrial DNA sequencing (1997) Sequencing of nuclear Neanderthal DNA would help human genome annotation and identify our most recent genetic differences Two groups used a bone found in 1980 in a cave in Croatia Carbon dated to 38,310 ± 2,130 years ago http://www.nature.com/nature/journal/v444/n7117/pdf/444254a.pdf

5. Contamination Challenge to find uncontaminated samples Tested >70 Neanderthal bone and tooth samples Most samples were too degraded or contaminated Six bones were further tested Amplified mtDNA that previous studies have shown to be different from modern humans Vi-80 bone (from Croatia) was best prospect for sequencing

6. Sequencing Approaches 454 sequencing: “sequencing by synthesis” Uses small fragments of DNA (advantageous) PCR every fragment to 10m copies Attach DNA to bead Solutions of nucleotides are progressively applied; photons are generated as each base is incorporated >1 million base pairs Metagenomic approach: Insert DNA fragments into plasmids Amplify in bacteria Sequence inserts using either pyrosequencing or Sanger method Can be used to target specific genes 65,000 base pairs

7. Vi-80 bone Recovered 254,933 unique sequences from Vi-80 bone (Nature group) Aligned sequences to complete genomes, environmental samples (env database), and GenBank set of redundant nucelotide sequences (nt)

8. Neanderthal chromosomes

9. Time of Divergence Science group: –used SNPs to approximate when modern humans and Neanderthals diverged –Estimated 120,000-670,000 years, with a best guess of 370,000 Nature group: –used PCR confirmed substitutions of mtDNA –assumed human-chimp divergence 4.7-8.4 millions years –estimate human-Neanderthal divergence of 461,000- 825,000 years; best guess of 516,000 when including nuclear DNA substitution information

10. Log-likelihood graphs

11. Nucleotide changes Generated 3-way alignments between Neanderthal, human, and chimp Estimate 7.1%-8.2% of sequence changes between human and chimp occurred after Neanderthal divergence

12. SNP study Compared Neanderthal sequences to human SNP sites Neanderthal has derived (human SNP) allele (as opposed to ancestral chimp allele) in 30% of all SNPs Possibly overestimation since SNPs analyzed are of high frequency in human and thus are likely older Estimate conflicts with their population split model Suggests interbreeding? Neanderthal X chromosome shows higher level of divergence than autosomes Possible gene flow from modern human males into Neanderthals

13. Ancestral Population Size Human effective population size is around 10,000 Chimps, gorillas, and orangutans have an effective population size 2-4 times larger Effective population size of human and chimp ancestor thought to be closer to apes than modern humans Estimate for Neanderthal is up to 12,000 with a best guess of 3,000 Indicates effective population size of hominids had decreased prior to human-Neanderthal split Dependent on what split time you use (time after which there was no more interbreeeding between groups)

14. Summary Papers describe sequencing of about.03% of Neanderthal autosomal genes Groups agree that human and Neanderthal genomes are <0.5% different Broad agreement on divergence time (370,000 vs 516,000) Possible gene flow between human and Neanderthal, but not definitive yet

15. Future Plans Is it possible to fully sequence genome? 1x coverage requires 20g of bone and 6000 runs on the 454 sequencing platform Nature authors think they can make the sequencing 10x more efficient Their goal is to fully sequence the Neanderthal genome within two years Currently 5 million bp are available Science authors are targeting FOXP2 gene –Linked to speech and language; Present in many animals, but human version differs from chimp

16. Happy Thanksgiving! 1-800-BUTTERBALL