Analysisofonemillionbasepairsof NeanderthalDNA

RichardE.Green,JohannesKrause,SusanE.Ptak,AdrianW.Briggs,MichaelT.Ronan,JanF.Simons,LeiDu, MichaelEgholm,JonathanM.Rothberg,MajaPaunovic&SvantePbo

NatureVol444|16November2006|doi:10.1038/nature05336

BrbaraVerduzco|JHU410.666GenomicSequencingandAnalysis|September2012

Introduction
Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. Neanderthals were first recognized as a distinct group of hominids from fossil remains discovered 150 years ago at Feldhofer in Neander Valley, outside Dsseldorf, Germany.

A Neanderthal nuclear genome sequence would be an invaluable resource for annotating the human genome to mark when remarkable morphological, behaviorural, and cognitive changes occurred.

Methodology
IdentificationofaNeanderthalfossilforDNAsequencing Avoid: Degradation Chemicaldamage DNAisirretrievableormisread Contamination specimens,labreagents,andinstruments

mtDNA
Hypervariable region

Picksample

PCR

Clone

454

BFX Analysis

More PCRs

More Analysis

Check for biomolecular preservation by using samples of a few milligrams for amino acid analysis. The vast majority of these samples had low overall contents of amino acids and/or high levels of amino acid racemization, a stereoisomeric structural change that affects amino acids in fossils, indicating that they are unlikely to contain retrievable endogenous DNA.
3
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Preparation of sample

PCR

Figure1 Ratio of Neanderthal to modern human mtDNA in six hominid fossils. For each fossil, primer pairs that amplify a long (119 base pairs; upper lighter bars) and hort (63 base pairs; lower darker bars) product were used to amplify segments of the mtDNA hypervariable region. The products were sequenced and determined to be either of Neanderthal (yellow) or modern human (blue) type.
4
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Sequencing Procedure and Methods

454

5
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Sequencing Procedure and Methods

454

Sequentialsinglenucleotideextension Threemajorsubsystems: fluidicassembly(A),a flowchamber CCDcamerabasedimagingassembly

6
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Overview of Results

Figure4 LocationonthehumankaryotypeofNeanderthalDNAsequences
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Overview of Results

Figure 5 Schematic tree illustrating the number of nucleotide changes inferred to have occurred on hominoid lineages.
8
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

Compare to other methods [ Illumina ]

Bridgeamplification Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.

9
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336

PresentationGuidelines:
Timeforpresentationwillbeapproximately20 minutesperstudent Eachstudentwillpresentasummaryofthe paperconsistingofthefollowing:
Principlesofthemethod(1slide) Preparationofsample(1slide) Sequencingprocedureandmethod(2slides) Overviewofresults(2slides) Comparebrieflytoothermethods(1slide)

Gettowork!

Afterthepresentation,emailtheinstructors yourslides!

10 BrbaraVerduzco|JHU410.666GenomicSequencingandAnalysis|September2012