Initial sequencing and analysis of the human genome - Vitagenes

articlesBy agreement among the centres, the collection of draft clonesproduced by each centre was required to have fourfold averagesequence coverage, with no clone below threefold. (For this purpose,sequence coverage was de®ned as the average number of timesthat each base was independently read with a base-quality scorecorresponding to at least 99% accuracy.) We attained an overallaverage of 4.5-fold coverage across the genome for draft clones. Afew of the sequenced clones fell below the minimum of threefoldsequence coverage or have not been formally designated by centresas meeting draft standards; these are referred to as predraft (Table 2).Some of these are clones that span remaining gaps in the draftgenome sequence and were in the process of being sequenced on 7October 2000; a few are old submissions from centres that are nolonger active.The lengths of the initial sequence contigs in the draft clones varyas a function of coverage, but half of all nucleotides reside in initialsequence contigs of at least 21.7 kb (see below). Various propertiesof the draft clones can be assessed from instances in which there wassubstantial overlap between a draft clone and a ®nished (or nearly®nished) clone. By examining the sequence alignments in theoverlap regions, we estimated that the initial sequence contigs in adraft sequence clone cover an average of about 96% of the clone andare separated by gaps with an average size of about 500 bp.Although the main emphasis was on producing a draft genomesequence, the centres also maintained sequence ®nishing activitiesduring this period, leading to a twofold increase in ®nishedsequence from June 1999 to June 2000 (Fig. 4). The total amountof human sequence in this ®nal form stood at more than 835 Mb on7 October 2000, or more than 25% of the human genome. Thisincludes the ®nished sequences of chromosomes 21 and 22 (refs 93,94). As centres have begun to shift from draft to ®nished sequencingin the last quarter of 2000, the production of ®nished sequence hasincreased to an annualized rate of 1 Gb per year and is continuing torise.Table 3 Total human sequence deposited in the HTGS division of GenBankSequencing centreTotal humansequence (kb)Finished humansequence (kb)Whitehead Institute, Center for Genome Research* 1,196,888 46,560The Sanger Centre* 970,789 284,353Washington University Genome Sequencing Center* 765,898 175,279US DOE Joint Genome Institute 377,998 78,486Baylor College of Medicine Human Genome Sequencing 345,125 53,418CenterRIKEN Genomic Sciences Center 203,166 16,971Genoscope 85,995 48,808GTC Sequencing Center 71,357 7,014Department of Genome Analysis, Institute of Molecular 49,865 17,788BiotechnologyBeijing Genomics Institute/Human Genome Center 42,865 6,297Multimegabase Sequencing Center; Institute for Systems 31,241 9,676BiologyStanford Genome Technology Center 29,728 3,530The Stanford Human Genome Center and Department of 28,162 9,121GeneticsUniversity of Washington Genome Center 24,115 14,692Keio University 17,364 13,058University of Texas Southwestern Medical Center at Dallas 11,670 7,028University of Oklahoma Advanced Center for Genome 10,071 9,155TechnologyMax Planck Institute for Molecular Genetics 7,650 2,940GBF ± German Research Centre for Biotechnology 4,639 2,338Cold Spring Harbor Laboratory Lita Annenberg Hazen 4,338 2,104Genome CenterOther 59,574 35,911Total 4,338,224 842,027.............................................................................................................................................................................Total human sequence deposited in GenBank by members of the International Human GenomeSequencing Consortium, as of 8 October 2000.The amount of total sequence (®nished plus draftplus predraft) is shown in the second column and the amount of ®nished sequence is shown inthe third column. Total sequence differs from totals in Tables 1 and 2 because of inclusion ofpadding characters and of some clones not used in assembly. HTGS, high throughput genomesequence.*These three centres produced an additional 2.4 Gb of raw plasmid paired-end reads (see Table 4),consisting of 0.99 Gb from Whitehead Institute, 0.66 Gb from The Sanger Centre and 0.75 Gb fromWashington University.In addition to sequencing large-insert clones, three centresgenerated a large collection of random raw sequence reads fromwhole-genome shotgun libraries (Table 4; ref. 98). These 5.77million successful sequences contained 2.4 Gb of high-qualitybases; this corresponds to about 0.75-fold coverage and would bestatistically expected to include about 50% of the nucleotides in thehuman genome (data available at http://snp.cshl.org/data). Theprimary objective of this work was to discover SNPs, by comparingthese random raw sequences (which came from different individuals)with the draft genome sequence. However, many of these rawsequences were obtained from both ends of plasmid clones andthereby also provided valuable `linking' information that was usedin sequence assembly. In addition, the random raw sequencesprovide sequence coverage of about half of the nucleotides not yetrepresented in the sequenced large-insert clones; these can be usedas probes for portions of the genome not yet recovered.Assembly of the draft genome sequenceWe then set out to assemble the sequences from the individual largeinsertclones into an integrated draft sequence of the humangenome. The assembly process had to resolve problems arisingfrom the draft nature of much of the sequence, from the variety ofclone sources, and from the high fraction of repeated sequences inthe human genome. This process involved three steps: ®ltering,layout and merging.The entire data set was ®ltered uniformly to eliminate contaminationfrom nonhuman sequences and other artefacts that had notalready been removed by the individual centres. (Information aboutcontamination was also sent back to the centres, which are updatingthe individual entries in the public databases.) We also identi®edinstances in which the sequence data from one BAC clone wassubstantially contaminated with sequence data from another(human or nonhuman) clone. The problems were resolved inmost instances; 231 clones remained unresolved, and these wereeliminated from the assembly reported here. Instances of lowerlevels of cross-contamination (for example, a single 96-well microplatemisassigned to the wrong BAC) are more dif®cult to detect;some undoubtedly remain and may give rise to small spurioussequence contigs in the draft genome sequence. Such issues arereadily resolved as the clones progress towards ®nished sequence,but they necessitate some caution in certain applications of thecurrent data.The sequenced clones were then associated with speci®c clones onthe physical map to produce a `layout'. In principle, sequencedclones that correspond to ®ngerprinted BACs could be directlyassigned by name to ®ngerprint clone contigs on the ®ngerprintbasedphysical map. In practice, however, laboratory mixups occasionallyresulted in incorrect assignments. To eliminate such problems,sequenced clones were associated with the ®ngerprint clonecontigs in the physical map by using the sequence data to calculate aTable 4 Plasmid paired-end readsTotal reads deposited* Read pairs² Size range of inserts(kb)Random-sheared 3,227,685 1,155,284 1.8±6Enzyme digest 2,539,222 761,010 0.8±4.7Total 5,766,907 1,916,294.............................................................................................................................................................................The plasmid paired-end reads used a mixture of DNA from a set of 24 samples from the DNAPolymorphism Discovery Resource (http://locus.umdnj.edu/nigms/pdr.html). This set of 24 anonymousUS residents contains samples from European-Americans, African-Americans, Mexican-Americans, Native Americans and Asian-Americans, although the ethnicities of the individualsamples are not identi®ed. Informed consent to contribute samples to the DNA PolymorphismDiscovery Resource was obtained from all 450 individuals who contributed samples. Samples fromthe European-American, African-American and Mexican-American individuals came from NHANES(http://www.cdc.gov/nchs/nhanes.htm); individuals were recontacted to obtain their consent forthe Resource project. New samples were obtained from Asian-Americans whose ancestry wasfrom a variety of East and South Asian countries. New samples were also obtained for the NativeAmericans; tribal permission was obtained ®rst, and then individual consents. See http://www.nhgri.nih.gov/Grant_info/Funding/RFA/discover_polymorphisms.html and ref. 98.*Re¯ects data deposited with and released by The SNP Consortium (see http://snp.cshl.org/data).² Read pairs represents the number of cases in which sequence from both ends of a genomiccloned fragment was determined and used in this study as linking information.868 © 2001 Macmillan Magazines Ltd NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com