Lecture Slides Lecture 2 Slides

7/30/2019 Lecture Slides Lecture 2 Slides

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TheHumanGenomeProject

HaroldRiethman,PhD

[email protected]


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TheHumanGenomeProject

HumanGenomeMapping

SangerDNASequencing

PublicConsortium

Draft

Sequence

CeleraDraftSequence

Analysis

of

Draft

Human

Genome

Sequence


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Landmarks FragmentEnds

DNAprobes Restriction

Enzyme

STS Meiosis

Radiation

Cloneends

HumanGenomeMapping


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MappingResolution

From

Matise

et

al.,

Genome

Analysis

Vol

4,

1999


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MeioticRecombinationCreatesthe

ChromosomeBreakpoints

for

Genetic

Linkage

Maps


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FromChakravarti and

Lynn,

Genome

Analysis

Vol 4,

1999


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Analysisof

Linkage

Data

Comparetheobservedfrequencythat2markersarecotransmittedtoprogenywiththeexpectedfrequencyofcotransmission

if

by

random

chance

Formarkersthatareclosetogetheronachromosomethe

ratioof

observed/expected

is

high,

said

to

be

linked

markers

Linkagemapdistancesarestatisticalestimatesofmarker

distancebased

on

recombination

frequencies

Forhumansthetotalnumberofbreaksandhencetheresolutionofhumangeneticlinkagemapsislimited(1cM,roughly

=1Mb)

because

the

number

of

meiosis

is

limited.


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RadiationCreates

the

Chromosome

Breaks

>radiation, >frequencyofbreaks

tightlylinkedmarkerswilltendtoberetainedonthe

sameRHfragments

RadiationHybrid

Maps


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Like linkage maps, RH maps are based upon probabilities

Statistical errorinmarkerorderanddistance

RetentionBiasnearCentromeres

MappingpanelsofDNAavailable

RadiationHybrid

Maps


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CloneLibraryPreparation YACs&BACs

CloneOverlapDetectionandContig

Construction STSandFingerprintMethods

Contig PlacementAlong

Chromosome

AlignmentwithSTSmaps&FISH

Clonebased

Physical

Maps


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A) Yeast Artificial Chromosome (YAC) 200 kb 2 Mb

B) BacterialArtificialChromosome(BAC) 150kb 250kb

C)

Cosmid/Fosmid 40kb

CloningSystems


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Cloneends

Clonebased

PhysicalMap

FromDunham

et

al.,

GenomeAnalysisVol

3,1999


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UsingthesamesetofSTSmarkerspermits

integrationofthedifferentmaps

YACbasedCloneMapswereusefulfor

preparing100kbresolutionSTSmaps,but

BACbasedclonemapsbecamethe

workhorsefor

sequencing


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HumanSequence

ready

Physical

Map


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BACclonebased

physicalmap

FromWaterston

et

al.,

PNAS

2002


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BACCloning

150 250kb

FromBirren etal.,

Genome

Analysis

Vol 3,

1999


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BAC

fingerprint

mapping

FromMarra

et

al.

Genome

Research

1997


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FromMarra et

al.

Genome

Research

1997


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SangerDNA

Sequencing


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FromShendureandJi,Nat.

Biotech2008

SangerDNAsequencing


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BACclonebased

physicalmap

FromWaterston

et

al.,

PNAS

2002

SangerDNA

Sequencing

Assembly

Anchoringto

Chromosome


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Preparationof

Templates

and

Sequencing

Reactions

highlyautomated

1.)Growthofclones

coloniespickedrobotically,transferredtosmallculturesin96wellformats

2.)PurificationofDNA

reagentsaddedandremovedrobotically,procedurescarriedoutin96wellformat

3)DNAsequencingreactions

reagentsadded

and

removed

robotically,

procedures

carried

out

in

96

well

or

96

x4(384

well)

format


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ProductionSequencingatMITsWhiteheadInstitute


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FromGordon

et

al.,

Genome

Research

1998.

SequenceAssembly


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Sequencing

BACAteachstep,sequencesdepositedintoGenBank withanAccessionnumber

a) 96reactions (Phase0)

samplesequences,

overlap

detection

b)35xcoverage (Phase1)

assembleddraft

c) 8 10xcoverage (Phase2)

Highqualitydraftassemblies

d) finishedsequence (Phase3)

qvalues

>40,

no

gaps

in

sequence


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Green,1997 WeberandMeyers,1997

FromWaterston

et

al.,

PNAS

2002


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InternationalHumanGenomeSequencingConsortium

DraftHuman

Genome

Sequence:

Nature

Feb.15,

2001

Goal:Immediateand

unrestrictedpublicaccessto

genomesequence


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BACclonebased

physicalmap

FromWaterston

et

al.,

PNAS

2002

SangerDNA

Sequencing

Assembly

Anchoringto

Chromosome

PublicDraftSequenceStrategy


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AssemblingtheDraftSequence

Filtering

Nonhumansequences

ContaminationwithotherBACsequences

Layout

Sequenceslayed overphysicalmap

Lab

mix

upsElectronicdigesttoplaceincorrectcontig

BACendsequences

STSandFISHmaps

Merging

GigAssembler

Sequencecontig scaffold


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FromIHGSC

Nature

2001


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N50Length

Maximumlength

Lsuch

that

50%

of

all

nucleotides

lieincontigs (orscaffolds)oflengthL


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FromIHGSCNature2001


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CeleraGenomicsDraftHumanGenomeSequence

ScienceFeb.16,

2001

Businessmodel:

patent

genesforcommercial

products,sellaccesstothe

genome

sequence

and

Celeragenerated

annotations


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Freepublicdatawasusedandincorporatedby

Celerainto

its

assembly

Summaryof

Input

Sequence:

1.) 15GbofCelerarawsequence5xcoverage

2.) 4.4Gb

of

Public

Draft

Sequence

(derivedfromabout23Gb(7.5xcoverage)

ofrawsequence)

a.)

shredded

to

a

perfect

2x

coverage

of

Bactigsb.)2.96xcoverageofgenome


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FromVenter

et

al.,

Science

2001

Celeraassemblystrategy


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FromVenteret

al.,Science

2001


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WGA:WholeGenomeAssembly

5xcelera reads+3xpublicfauxreads

+celera mate

pair

data

TrueWGA(?)


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BACtigs

are

Mapped

BAC

sequence

contigs from

the

publicproject

CSA:CompartmentalizedShotgunAssembly

(1) Bactigs fromregion+celera readsmatchingBactigs

(2) Celerauniquescaffoldsmappingtoregion

(3)scaffoldtilingforcompartmentcheckedmanually

(4)Publicsequencewithincompartmentshredded,thenreassembled

withcelera reads

from

compartment


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FromVenteret

al.,Science

2001

Comparison Public Draft View


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Comparison,PublicDraftView


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Comparison,CeleraView


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Analysis

of

Draft

Genome

Sequences


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Public

Data

NationalCenterforBiotechnologyInformationwww.ncbi.nlm.nih.gov/genome/guide/human/

UCSCGenomeBrowser extensiveannotationgenome.ucsc.edu/

Footnote:Initially,

Celera

data

was

available

only

through

licensingagreements,butlateritwasdepositedintothepublic

databases


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Human

Genome

Size:

~3billion

base

pairs

No.ofgenes:~40,000

Average

gene

density:

12

genes/

Mb Generichchromosome:17,19,22

Genepoorchromosome:4,13,18,X,Y


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CpG

islands RegionswithhigherfrequencyofCpGdinucleotides.

>

200

bp

regions

with

>

50%

GC. Associatedwith5endsofgenes/neartranscriptionalstart

sites.

30,000 50,000inthehumangenome.

Y:2.9islands/Mb; 19:43islands/Mb

GoodcorrelationofgenedensityandCpGislands


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Recombination

Rate

Onaverage: higherinfemalesthanmales.

Highlyvariableamongdifferentgenomic

regions.

Higher telomericregions.

Lower aroundthecentromeres.

Repeat Content


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RepeatContent

About50%ofthegenome

5classesofrepetitiveelements: transposonderivedrepeats(interspersedrepeats) 45%

genome;LINEs,SINEs,LTRs,DNAtransposons

inactiveretroposed copiesofcellulargenes(pseudogenes e.g.intronless inactivatedgenes)

simplesequence

repeats

micro

,minisatellites

segmentalduplications

blocksoftandemly repeatedsequences(e.g.aroundcentromeres,telomeres,shortarmsofacrocentricchromosomes)


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Sequence

Variation Unrelatedindividualsare99.9%identicalat

the

DNA

sequence. Mostcommontypeofvariant=single

nucleotidepolymorphisms(SNPs)

GGATCTA GGAGCTACCTAGAT CCTCGAT

SNPrate

~1per

1,200

bp;

1%

of

them

affectproteinfunction.


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Gene

Prediction

Methods DirectevidenceoftranscriptionprovidedbyESTs

ormRNA.

Indirectevidenceofsequencesimilaritytoknown

genesand

proteins.

Abinitio recognitionofexonsusingHMMs Genescan,

Genie.

Estimated gene number from the draft


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Estimatedgenenumberfromthedraft

sequences:

35,000to40,000

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Lecture Slides Lecture 2 Slides