(2.5.11)--finishing_the_euchromatic_sequen.pdf

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1、 2004 Nature PublishingGroupFinishing the euchromatic sequence ofthe human genomeInternational Human Genome Sequencing Consortium*A list of authors and their affiliations appears in the Supplementary Information.The sequence of the human genome encodes the genetic instructions for human physiology,a

2、s well as rich information abouthuman evolution.In 2001,the International Human Genome Sequencing Consortium reported a draft sequence of the euchromaticportion of the human genome.Since then,the international collaborationhas worked to convert this draftinto a genome sequencewith high accuracy and

3、nearly complete coverage.Here,we report the result of this finishing process.The current genomesequence(Build35)contains2.85billionnucleotidesinterruptedbyonly341gaps.Itcovers,99%oftheeuchromaticgenomeandisaccuratetoanerrorrateof,1eventper100,000bases.Manyoftheremainingeuchromaticgapsareassociatedwi

4、thsegmentalduplications and will require focused work with new methods.The near-complete sequence,the first for a vertebrate,greatlyimprovestheprecisionofbiologicalanalysesofthehumangenomeincludingstudiesofgenenumber,birthanddeath.Notably,thehuman enome seems to encode only 20,00025,000 protein-codi

5、ng genes.The genome sequence reported here should serve as afirm foundation for biomedical research in the decades ahead.The Human Genome Project(HGP)was launched in 1990 with thegoal of obtaining a highly accurate sequence of the vast majority ofthe euchromatic portion of the human genome.The initi

6、al workfollowed a two-pronged approach:(1)the mapping of the humanand mouse genomes19to allow the study of inherited disease andprovide a crucial scaffold for genome assembly;and(2)thesequencing of organisms with smaller,simpler genomes1014toserveasatestbedfor methoddevelopmentandassistininterpretin

7、gthe human genome.With success along both paths,the sequencingof the human genome itself eventually became feasible.The Inter-national Human Genome Sequencing Consortium(IHGSC),anopen collaboration involving twenty centres in six countries,wasformed to carry out this component of the HGP.In February

8、 2001,the IHGSC15and Celera Genomics16eachreported draft sequences providing a first overall view of thehuman genome.These sequences allowed systematic study of thehuman genome itself,including identification of genes,combina-torial architecture of proteins,regional differences in genomecomposition,

9、distribution and history of transposable elements,distribution of polymorphism and relationship between geneticrecombination and physical distance.Moreover,systematic knowl-edge of the human genome has enabled new tools and approachesthat have markedly accelerated biomedical research.Bothdraftsequen

10、ces,however,hadimportantshortcomings.TheIHGSC sequence,for example,omitted,10%of the euchromaticgenome;it was interrupted by,150,000 gaps;and the order andorientation of many segments within local regions had not beenestablished.The IHGSC thus turned to the challenge of completingthe sequence of the

11、 euchromatic genome.Operationally,a finishedsequence was defined as having an error rate of,at most,one eventper 104bases,and the goal for completion was coverage in finishedsequence of at least 95%of the euchromatic genome,with the onlygaps being those refractory to all available techniques17(see h

12、ttp:/www.genome.gov/10000923).The goal was challenging because thehumangenomeisrepletewithsuchfeaturesasdispersedrepeatsandlarge segmental duplications,which greatly complicate the deter-mination of genome structure and sequence.In fact,near-completesequences have been obtained so far only for three

13、 multicellularorganisms:thenematode13,mustardweed18andthefruitfly19.Thesegenomes are all roughly 30-fold smaller than the human genomeand have much simpler structure.We describe here the results of a multiyear effort by the IHGSCtowards the goal of a complete human sequence.The number ofgaps has bee

14、n reduced 400-fold to only 341,most of which areassociated with segmental duplications and will require newmethods for resolution.The assembled near-complete genomesequence has an error rate of only,1 event per 100,000 bases;itcontains 2.85 billion nucleotides and covers,99%of the euchro-matic genom

15、e.This paper describes the current genome sequenceand the process used to produce it;examines the accuracy andcompleteness of the sequence;and illustrates biological analysesmade possible by the sequence.We do not attempt here a compre-hensive analysis of the contents of the human genome.An initiala

16、nalysis was previously reported15and a series of papers is beingwritten describing the individual chromosomes17,2030,includingannotation of genes and other features.Current genome sequenceFinishing processThe process of converting the initial draft sequence into a near-complete sequence is referred

17、to as finishing.It is a complexiterative process that proceeds simultaneously at multiple scales,ranging from single nucleotides to the integrity of whole chromo-somes.The fundamental challenge is that genomic regions that arenot well represented or readily resolved through random shotgunsequencing

18、tend to be highly enriched in problematic sequences.Resolving such regions required the development of specialapproaches,which evolved substantially over time and variedamong centres.Broadly,the finishing process involved two distinct components:(1)producingfinished maps,consistingofcontinuousandacc

19、uratepaths of overlapping large-insert clones spanning the euchromaticregionofeachchromosomearm;and(2)producingfinishedclones,consisting of continuous and accurate nucleotide sequence acrosseach large-insert clone.In practice,these two components weretightly intertwined in that progress in each ofte

20、n depended onresultsfromtheother.ThecomponentsaredescribedinBoxes1and2.Further information about the finishing process and finishingstandardscan befound in the Supplementary Information(Note 1)and at http:/www.genome.gov/10000923.In total,we generated a shotgun sequence from 59,208 large-insert clon

21、es(total length,5.84 gigabases(Gb)and finished thesequence from 45,742 of these clones(total length,3.67Gb).Theclones consisted primarily of bacterial artificial chromosomesarticlesNATURE|VOL 431|21 OCTOBER 2004| 2004 Nature PublishingGroup(BACs),but also included some P1-artificial chromosomes(PACs

22、),yeast artificial chromosomes(YACs),fosmids and cosmids;theycarried DNA from multiple anonymous sources15.We then chose aclone tiling path of 26,720 overlapping clones across the genome,selected a sequence tiling path of directly adjacent,non-overlappingsegments from consecutive clones and concaten

23、ated these segmentsto create a near-complete genome sequence.Contributions of theIHGSC centres to this finishing phase are shown in Table 1.Genome sequenceThe human sequence reported here consists of 2,851,330,913nucleotides,lying almost entirely within the euchromatic portionof the genome(Table 2).

24、Itis interrupted byonly 341 gaps,of which33 gaps(totalling,198 megabases(Mb)reflect heterochromatin,which was not targeted by the HGP,and 308 gaps(totalling,28Mb)are euchromatic.The euchromatic genome is thus,2.88Gb and the overall human genome is,3.08Gb.The long-range continuity of the current geno

25、me sequence is high by variousmeasures(Table 3).The N50 length is 38.5Mb and the N-averagelength is 40.9Mb;these values are,1,000-fold larger than the sizeof a typical human gene.(The first statistic is the length x such thatatleast50%ofnucleotideslieinacontinuoussegmentoflength$x,whereas the second

26、 is the average length of the contiguous segmentcontaining a randomly chosen nucleotide.)Focusing on individualBox 1Finishing the physical mapThe hierarchical strategy used two kinds of genome maps as afoundation for producing finished sequence:sequence-tagged site(STS)maps6and clone maps7.The first

27、 provided global landmarks bypositioning tens of thousands of STSs through genetic mapping,radiation hybrid mapping and STS-content mapping.The secondprovided overlapping clones for sequencing and was obtained bycomparing restriction-digest fingerprints of hundreds of thousands ofBAC clones to creat

28、e local contigs.The two maps were integrated byanchoring the contigs to the global landmarks.The initial random phase generated a physical map covering 9698%of the euchromatic genome in,1,000 anchored contigs separated bygaps with average size of,100kb7.Overlapping clones were chosenfrom this map to

29、 produce the draft sequence.The subsequent finishingphase involved verifying clone overlaps and closing gaps in the initialmap.The finishing process for each chromosome was overseen by adesignated centre and managed by a dedicated coordinator,whointegrated and reviewed information from contributing

30、centres.Clone overlaps were verified by analysing finished sequence toconfirm that the terminal sequence overlapped for$2kb with$99.6%identity.(Perfect identity was not expected in all cases,because theclones might derive from different haplotypes and thus differ atpolymorphic sites.)A small number

31、of overlaps(n 308)falling belowthis threshold were accepted,because the sequencing centrepresented additional evidence that the overlap was correct.Forexample,smaller overlaps might be confirmed by a partiallysequenced clone spanning the overlap,or a region of high variationmight be shown to be due

32、to allelic variation.All these exceptions arerecorded in electronic tags shown on the UCSC genome browser.Remaining overlaps were rejected and counted as new gaps in themap.Closing gaps required identifying a tiling path of clones spanning theregion.Methods used are summarized in Box 1 Fig.1.The pri

33、maryapproach was iterative walking from the ends of contigs:a stretch ofterminalsequencefromaterminalclonewasusedtoidentifyanewcloneextendingthecontig.Theprocedurewasrepeateduntiliteitheryieldedaclone connecting to the neighbouring contig or reached a dead end.Walking used both experimental(hybridiz

34、ation to arrayed BAC libraries)and computational(comparison with a database of BAC end-sequences)methods to identify candidate clones.Also,it wassometimes possible to parachute into gaps by identifying sequenceslikely to lie within the gap(such as STSs,mRNAs and mouse sequencesin regions of conserve

35、d synteny)and usingthem as hybridization probesfor BACs.Some gaps could not be closed,either because no new clone couldbe found despite screening of diverse and deep libraries(.30-foldphysicalcoverage)or becauseacomplexcollectionofcloneswasfoundwhose relationship proved impossible to discern owing t

36、o the presenceof extensive highly repetitive sequences or near-exact segmentalduplication.Telomeric regions of chromosomes were obtained by usinga specialized cloning system(half-YACs vectors)in which successfulpropagation required that the human insert DNA provided telomericfunction.By far,the most

37、 difficult regions of the genome were thosecontaining near-exact segmental duplications.A particularly challengingexample is shown in Box 1 Fig.2.Box 1 Figure 1 Simplified flowchart for finishing the physical map.Box 1 Figure 2 Illustration of a challenging region on chromosome Y.a,The sequenceorgan

38、ization of the palindromic repeat P3 is represented by the horizontal bar at the baseof the triangle.Arrows indicate orientation of sequences in the 283-kb arms of thepalindrome(b1,t1 on left and t2,b2 on right).A non-repeated 170-kb spacer(white)separates the arms.Above the horizontal bar,each dot

39、represents a perfect match of500bp.The near identity between the arms(99.94%)appears as a vertical line of dots,highlightedbythebluediamond.b,TilingpathofBACsfromRPCI-11library.c,Sequencedifferences,numbered 17,between arms of P3.Differences 3,5,6 and 7 are singlenucleotide differences,whereas 1,2,a

40、nd 4 are differences in the lengths of simpletandem repeats(microsatellites);L,long variant;S,short variant.These differencesallowed assignment of BACs to the correct arm of P3.articlesNATURE|VOL 431|21 OCTOBER 2004| 2004 Nature PublishingGroupchromosome arms,the N50 length exceeds half the length o

41、f thearm in three-quarters of cases(Table 3).The sequence is denoted as NCBI Human Build 35(May 2004),with the individual chromosomes having accession numbersNC000001 to NC000024(see Supplementary Information Note 3concerning additional sequence data).The analyses reported herewere performed on Buil

42、d 35 or,in a few cases,its immediatepredecessor,Build 34(which differed only slightly).The posteraccompanying this paper displays the 24 human chromosomes,together with various biological annotations.These include GCcontent,repeat content,segmental duplications,protein-codinggenes,sequence similarit

43、y and synteny conservation with mouse,sequence similarity with the pufferfish,and density of single-nucleo-tide polymorphisms in the human genome.Many additional anno-tations can be found on public genome browsers(http:/genome.ucsc.edu/;http:/www.ensembl.org/;http:/www.ncbi.nlm.nih.gov/genome/guide/

44、human/),which are regularly updated.Comparison with draft sequenceThe near-complete sequenceis a great improvementover the earlierdraft sequence.It has substantially fewer gaps(341 versus 147,821)and greater continuity(38,500 kilobases(kb)versus 81kb for N50contig size),reflecting an overall improve

45、ment of,475-fold.Thedraft sequence contained regions in which the local order andorientation were unknown;these have now been resolved.Thecase of chromosome 7 is illustrated in Fig.1.Additionally,the draftsequence contained substantial artefactual duplication,includinglocal events caused by errors i

46、n merging some adjacent BAC-basedsequences,made by the first-generation global assembly program,andglobaleventscausedbycontaminationofshotgunassembliesofsome BACs with data from other clones.These artefacts have nowbeen eliminated.Accuracy and completenessBecause the human genome sequence is intende

47、d to serve as aTable 1 Bases sequenced in Build 35CentreFinished sequence totals(kb)Finished sequence in Build 35(kb).SC919,388849,650WUGSC645,062583,032WIBR562,096373,760JGI/SHGC485,085313,988BCM320,735280,963RIKEN155,769112,047UWGC145,745105,573GS99,97078,467GTC45,71032,972UWMSC39,22728,367Keio44,

48、90520,780IMB73,67720,053Beijing38,07917,114MPIMG9,8385,673GBF8,3255,547UOKNOR18,6575,311TIGR10,3903,054CGM2,7681,766SDSTDC7,7921,403UTSW8,555196Other30,74511,621Total3,672,5162,851,336.Columns indicate total finished sequence deposited in public databases(including overlaps andalternative alleles)an

49、d finished sequence incorporated into Build 35(consisting of those cloneschosenforinclusioninthetilingpathbytheindividualchromosomecoordinators).Thetotalincludesfinished sequence completed at the time of the draft sequence15,17,20and subsequently.Someclonesweresequencedtodraftcoveragebyonecentre,the

50、nfinishedbyanother.Abbreviations:SI,Wellcome Trust Sanger Institute(Hinxton,UK);WUGSC,Washington University Genome Sequen-cing Center(St Louis,USA);JGI,US Department of Energy Joint Genome Institute(Walnut Creek,USA);WIBR,Whitehead Institute for Biomedical Research(Cambridge,USA);BCM,BaylorCollegeof