NeanderthalsNoonan, 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

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/

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

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

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

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

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)

Neanderthal chromosomes

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

Log-likelihood graphs

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

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

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)

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

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

Happy Thanksgiving!

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