Initial sequencing and analysis of the human genome - Vitagenes

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articleswell as targeted regions of mammalian genomes 34±37 . These projectsshowed that large-scale sequencing was feasible and developed thetwo-phase paradigm for genome sequencing. In the ®rst, `shotgun',phase, the genome is divided into appropriately sized segments andeach segment is covered to a high degree of redundancy (typically,eight- to tenfold) through the sequencing of randomly selectedsubfragments. The second is a `®nishing' phase, in which sequencegaps are closed and remaining ambiguities are resolved throughdirected analysis. The results also showed that complete genomicsequence provided information about genes, regulatory regions andchromosome structure that was not readily obtainable from cDNAstudies alone.In 1995, genome scientists considered a proposal 38 that wouldhave involved producing a draft genome sequence of the humangenome in a ®rst phase and then returning to ®nish the sequence ina second phase. After vigorous debate, it was decided that such aplan was premature for several reasons. These included the need ®rstto prove that high-quality, long-range ®nished sequence could beproduced from most parts of the complex, repeat-rich humangenome; the sense that many aspects of the sequencing processwere still rapidly evolving; and the desirability of further decreasingcosts.Instead, pilot projects were launched to demonstrate the feasibilityof cost-effective, large-scale sequencing, with a target completiondate of March 1999. The projects successfully produced®nished sequence with 99.99% accuracy and no gaps 39 . They alsointroduced bacterial arti®cial chromosomes (BACs) 40 , a new largeinsertcloning system that proved to be more stable than the cosmidsand yeast arti®cial chromosomes (YACs) 41 that had been usedpreviously. The pilot projects drove the maturation and convergenceof sequencing strategies, while producing 15% of the humangenome sequence. With successful completion of this phase, thehuman genome sequencing effort moved into full-scale productionin March 1999.The idea of ®rst producing a draft genome sequence was revivedat this time, both because the ability to ®nish such a sequence was nolonger in doubt and because there was great hunger in the scienti®ccommunity for human sequence data. In addition, some scientistsfavoured prioritizing the production of a draft genome sequenceover regional ®nished sequence because of concerns about commercialplans to generate proprietary databases of human sequencethat might be subject to undesirable restrictions on use 42±44 .The consortium focused on an initial goal of producing, in a ®rstproduction phase lasting until June 2000, a draft genome sequencecovering most of the genome. Such a draft genome sequence,although not completely ®nished, would rapidly allow investigatorsto begin to extract most of the information in the human sequence.Experiments showed that sequencing clones covering about 90% ofthe human genome to a redundancy of about four- to ®vefold (`halfshotgun'coverage; see Box 1) would accomplish this 45,46 . The draftgenome sequence goal has been achieved, as described below.The second sequence production phase is now under way. Itsaims are to achieve full-shotgun coverage of the existing clonesduring 2001, to obtain clones to ®ll the remaining gaps in thephysical map, and to produce a ®nished sequence (apart fromregions that cannot be cloned or sequenced with currently availabletechniques) no later than 2003.Strategic issuesHierarchical shotgun sequencingSoon after the invention of DNA sequencing methods 47,48 , theshotgun sequencing strategy was introduced 49±51 ; it has remainedthe fundamental method for large-scale genome sequencing 52±54 forthe past 20 years. The approach has been re®ned and extended tomake it more ef®cient. For example, improved protocols forfragmenting and cloning DNA allowed construction of shotgunlibraries with more uniform representation. The practice of sequencingfrom both ends of double-stranded clones (`double-barrelled'shotgun sequencing) was introduced by Ansorge and others 37 in1990, allowing the use of `linking information' between sequencefragments.The application of shotgun sequencing was also extended byapplying it to larger and larger DNA moleculesÐfrom plasmids(, 4 kilobases (kb)) to cosmid clones 37 (40 kb), to arti®cial chromosomescloned in bacteria and yeast 55 (100±500 kb) and bacterialgenomes 56 (1±2 megabases (Mb)). In principle, a genome of arbitrarysize may be directly sequenced by the shotgun method,provided that it contains no repeated sequence and can be uniformlysampled at random. The genome can then be assembledusing the simple computer science technique of `hashing' (in whichone detects overlaps by consulting an alphabetized look-up table ofall k-letter words in the data). Mathematical analysis of theexpected number of gaps as a function of coverage is similarlystraightforward 57 .Practical dif®culties arise because of repeated sequences andcloning bias. Small amounts of repeated sequence pose littleproblem for shotgun sequencing. For example, one can readilyassemble typical bacterial genomes (about 1.5% repeat) or theeuchromatic portion of the ¯y genome (about 3% repeat). Bycontrast, the human genome is ®lled (. 50%) with repeatedsequences, including interspersed repeats derived from transposableelements, and long genomic regions that have been duplicated intandem, palindromic or dispersed fashion (see below). Theseinclude large duplicated segments (50±500 kb) with high sequenceidentity (98±99.9%), at which mispairing during recombinationcreates deletions responsible for genetic syndromes. Such featurescomplicate the assembly of a correct and ®nished genome sequence.There are two approaches for sequencing large repeat-richgenomes. The ®rst is a whole-genome shotgun sequencingapproach, as has been used for the repeat-poor genomes of viruses,bacteria and ¯ies, using linking information and computationalGenomic DNABAC libraryOrganizedmapped largeclone contigsBAC to besequencedShotgunclonesShotgunsequenceAssemblyHierarchical shotgun sequencing...ACCGTAAATGGGCTGATCATGCTTAAATGATCATGCTTAAACCCTGTGCATCCTACTG......ACCGTAAATGGGCTGATCATGCTTAAACCCTGTGCATCCTACTG...Figure 2 Idealized representation of the hierarchical shotgun sequencing strategy. Alibrary is constructed by fragmenting the target genome and cloning it into a largefragmentcloning vector; here, BAC vectors are shown. The genomic DNA fragmentsrepresented in the library are then organized into a physical map and individual BACclones are selected and sequenced by the random shotgun strategy. Finally, the clonesequences are assembled to reconstruct the sequence of the genome.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd863
articlesanalysis to attempt to avoid misassemblies. The second is the`hierarchical shotgun sequencing' approach (Fig. 2), also referredto as `map-based', `BAC-based' or `clone-by-clone'. This approachinvolves generating and organizing a set of large-insert clones(typically 100±200 kb each) covering the genome and separatelyperforming shotgun sequencing on appropriately chosen clones.Because the sequence information is local, the issue of long-rangemisassembly is eliminated and the risk of short-range misassemblyis reduced. One caveat is that some large-insert clones may sufferrearrangement, although this risk can be reduced by appropriatequality-control measures involving clone ®ngerprints (see below).The two methods are likely to entail similar costs for producing®nished sequence of a mammalian genome. The hierarchicalapproach has a higher initial cost than the whole-genome approach,owing to the need to create a map of clones (about 1% of the totalcost of sequencing) and to sequence overlaps between clones. Onthe other hand, the whole-genome approach is likely to requiremuch greater work and expense in the ®nal stage of producing a®nished sequence, because of the challenge of resolving misassemblies.Both methods must also deal with cloning biases, resulting inunder-representation of some regions in either large-insert orsmall-insert clone libraries.There was lively scienti®c debate over whether the humangenome sequencing effort should employ whole-genome or hierarchicalshotgun sequencing. Weber and Myers 58 stimulated thesediscussions with a speci®c proposal for a whole-genome shotgunapproach, together with an analysis suggesting that the methodcould work and be more ef®cient. Green 59 challenged these conclusionsand argued that the potential bene®ts did not outweigh thelikely risks.In the end, we concluded that the human genome sequencingeffort should employ the hierarchical approach for several reasons.First, it was prudent to use the approach for the ®rst project tosequence a repeat-rich genome. With the hierarchical approach, theultimate frequency of misassembly in the ®nished product wouldprobably be lower than with the whole-genome approach, in whichit would be more dif®cult to identify regions in which the assemblywas incorrect.Second, it was prudent to use the approach in dealing with anoutbred organism, such as the human. In the whole-genome shotgunmethod, sequence would necessarily come from two differentcopies of the human genome. Accurate sequence assembly could becomplicated by sequence variation between these two copiesÐbothSNPs (which occur at a rate of 1 per 1,300 bases) and larger-scalestructural heterozygosity (which has been documented in humanchromosomes). In the hierarchical shotgun method, each largeinsertclone is derived from a single haplotype.Third, the hierarchical method would be better able to deal withinevitable cloning biases, because it would more readily allowtargeting of additional sequencing to under-represented regions.And fourth, it was better suited to a project shared among membersof a diverse international consortium, because it allowed work andresponsibility to be easily distributed. As the ultimate goal hasalways been to create a high-quality, ®nished sequence to serve as afoundation for biomedical research, we reasoned that the advantagesof this more conservative approach outweighed the additionalcost, if any.A biotechnology company, Celera Genomics, has chosen toincorporate the whole-genome shotgun approach into its ownefforts to sequence the human genome. Their plan 60,61 uses amixed strategy, involving combining some coverage with wholegenomeshotgun data generated by the company together with thepublicly available hierarchical shotgun data generated by the InternationalHuman Genome Sequencing Consortium. If the rawsequence reads from the whole-genome shotgun component aremade available, it may be possible to evaluate the extent to which thesequence of the human genome can be assembled without the needfor clone-based information. Such analysis may help to re®nesequencing strategies for other large genomes.Technology for large-scale sequencingSequencing the human genome depended on many technologicalimprovements in the production and analysis of sequence data. Keyinnovations were developed both within and outside the HumanGenome Project. Laboratory innovations included four-colour¯uorescence-based sequence detection 62 , improved ¯uorescentdyes 63±66 , dye-labelled terminators 67 , polymerases speci®callydesigned for sequencing 68±70 , cycle sequencing 71 and capillary gelelectrophoresis 72±74 . These studies contributed to substantialimprovements in the automation, quality and throughput ofcollecting raw DNA sequence 75,76 . There were also importantadvances in the development of software packages for the analysisof sequence data. The PHRED software package 77,78 introduced theconcept of assigning a `base-quality score' to each base, on the basisof the probability of an erroneous call. These quality scores make itpossible to monitor raw data quality and also assist in determiningwhether two similar sequences truly overlap. The PHRAP computerpackage (http://bozeman.mbt.washington.edu/phrap.docs/phrap.html) then systematically assembles the sequence data using thebase-quality scores. The program assigns `assembly-quality scores'to each base in the assembled sequence, providing an objectivecriterion to guide sequence ®nishing. The quality scores were basedon and validated by extensive experimental data.Another key innovation for scaling up sequencing was thedevelopment by several centres of automated methods for samplepreparation. This typically involved creating new biochemicalprotocols suitable for automation, followed by construction ofappropriate robotic systems.Coordination and public data sharingThe Human Genome Project adopted two important principleswith regard to human sequencing. The ®rst was that the collaborationwould be open to centres from any nation. Although potentiallyless ef®cient, in a narrow economic sense, than a centralizedapproach involving a few large factories, the inclusive approachwas strongly favoured because we felt that the human genomesequence is the common heritage of all humanity and the workshould transcend national boundaries, and we believed thatscienti®c progress was best assured by a diversity of approaches.The collaboration was coordinated through periodic internationalmeetings (referred to as `Bermuda meetings' after the venue of the®rst three gatherings) and regular telephone conferences. Work wasshared ¯exibly among the centres, with some groups focusing onparticular chromosomes and others contributing in a genome-widefashion.The second principle was rapid and unrestricted data release. Thecentres adopted a policy that all genomic sequence data should bemade publicly available without restriction within 24 hours ofassembly 79,80 . Pre-publication data releases had been pioneered inmapping projects in the worm 11 and mouse genomes 30,81 and wereprominently adopted in the sequencing of the worm, providing adirect model for the human sequencing efforts. We believed thatscienti®c progress would be most rapidly advanced by immediateand free availability of the human genome sequence. The explosionof scienti®c work based on the publicly available sequence data inboth academia and industry has con®rmed this judgement.Generating the draft genome sequenceGenerating a draft sequence of the human genome involved threesteps: selecting the BAC clones to be sequenced, sequencing themand assembling the individual sequenced clones into an overall draftgenome sequence. A glossary of terms related to genome sequencingand assembly is provided in Box 1.The draft genome sequence is a dynamic product, which isregularly updated as additional data accumulate en route to the864 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com© 2001 Macmillan Magazines Ltd
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Initial sequencing and analysis of the human genome - Vitagenes

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