Initial sequencing and analysis of the human genome - Vitagenes

articlescontigs in 942 ®ngerprint clone contigs.The hierarchy of contigs is summarized in Fig. 7. Initial sequencecontigs are integrated to create merged sequence contigs, which arethen linked to form sequence-contig scaffolds. These scaffolds residewithin sequenced-clone contigs, which in turn reside within ®ngerprintclone contigs.The draft genome sequenceThe result of the assembly process is an integrated draft sequence ofthe human genome. Several features of the draft genome sequenceare reported in Tables 5±7, including the proportion represented by®nished, draft and predraft categories. The Tables also show thenumbers and lengths of different types of contig, for each chromosomeand for the genome as a whole.The contiguity of the draft genome sequence at each level is animportant feature. Two commonly used statistics have signi®cantdrawbacks for describing contiguity. The `average length' of a contigis de¯ated by the presence of many small contigs comprising only asmall proportion of the genome, whereas the `length-weightedaverage length' is in¯ated by the presence of large segments of®nished sequence. Instead, we chose to describe the contiguity as aproperty of the `typical' nucleotide. We used a statistic called the`N50 length', de®ned as the largest length L such that 50% of allnucleotides are contained in contigs of size at least L.The continuity of the draft genome sequence reported here andthe effectiveness of assembly can be readily seen from the following:half of all nucleotides reside within an initial sequence contig of atleast 21.7 kb, a sequence contig of at least 82 kb, a sequence-contigscaffold of at least 274 kb, a sequenced-clone contig of at least 826 kband a ®ngerprint clone contig of at least 8.4 Mb (Tables 6, 7). Thecumulative distributions for each of these measures of contiguityare shown in Fig. 8, in which the N50 values for each measure can beseen as the value at which the cumulative distributions cross 50%.We have also estimated the size of each chromosome, by estimatingthe gap sizes (see below) and the extent of missing heterochromaticsequence 93,94,105±108 (Table 8). This is undoubtedly an oversimpli®cationand does not adequately take into account the sequence statusof each chromosome. Nonetheless, it provides a useful way to relatethe draft sequence to the chromosomes.Quality assessmentThe draft genome sequence already covers the vast majority of thegenome, but it remains an incomplete, intermediate product that isregularly updated as we work towards a complete ®nished sequence.The current version contains many gaps and errors. We thereforesought to evaluate the quality of various aspects of the current draftgenome sequence, including the sequenced clones themselves, theirassignment to a position in the ®ngerprint clone contigs, and theassembly of initial sequence contigs from the individual clones intosequence-contig scaffolds.Nucleotide accuracy is re¯ected in a PHRAP score assigned toeach base in the draft genome sequence and available to usersthrough the Genome Browsers (see below) and public databaseentries. A summary of these scores for the un®nished portion of thegenome is shown in Table 9. About 91% of the un®nished draftgenome sequence has an error rate of less than 1 per 10,000 bases(PHRAP score . 40), and about 96% has an error rate of less than 1in 1,000 bases (PHRAP . 30). These values are based only on thequality scores for the bases in the sequenced clones; they do notre¯ect additional con®dence in the sequences that are represented inoverlapping clones. The ®nished portion of the draft genomesequence has an error rate of less than 1 per 10,000 bases.Individual sequenced clones. We assessed the frequency of misassemblies,which can occur when the assembly program PHRAPjoins two nonadjacent regions in the clone into a single initialsequence contig. The frequency of misassemblies depends heavilyon the depth and quality of coverage of each clone and the nature ofthe underlying sequence; thus it may vary among genomic regionsand among individual centres. Most clone misassemblies are readilycorrected as coverage is added during ®nishing, but they may havebeen propagated into the current version of the draft genomesequence and they justify caution for certain applications.We estimated the frequency of misassembly by examininginstances in which there was substantial overlap between a draftclone and a ®nished clone. We studied 83 Mb of such overlaps,involving about 9,000 initial sequence contigs. We found 5.3instances per Mb in which the alignment of an initial sequencecontig to the ®nished sequence failed to extend to within 200 basesTable 6 Clone level contiguity of the draft genome sequenceChromosome Sequenced-clone contigs Sequenced-clone-contig scaffolds Fingerprint clone contigs with sequenceNumber N50 length (kb) Number N50 length (kb) Number N50 length (kb)All 4,884 826 2,191 2,279 942 8,3981 453 650 197 1,915 106 3,5372 348 1,028 127 3,140 52 10,6283 409 672 201 1,550 73 5,0774 384 606 163 1,659 41 6,9185 385 623 164 1,642 48 5,7476 292 814 98 3,292 17 24,6807 224 1.074 86 3,527 29 20,4018 292 542 115 1,742 43 6,2369 143 1,242 78 2,411 21 29,10810 179 1,097 105 1,952 16 30,28411 224 887 89 3,024 31 9,41412 196 1,138 76 2,717 28 9,54613 128 1,151 56 3,257 13 25,25614 54 3,079 27 8,489 14 22,12815 123 797 56 2,095 19 8,27416 159 620 92 1,317 57 2,71617 138 831 58 2,138 43 2,81618 137 709 47 2,572 24 4,88719 159 569 79 1,200 51 1,53420 42 2,318 20 6,862 9 23,48921 5 28,515 5 28,515 5 28,51522 11 23,048 11 23,048 11 23,048X 325 572 181 1,082 143 1,436Y 27 1,539 20 3,290 8 5,135UL 47 227 40 281 40 281...................................................................................................................................................................................................................................................................................................................................................................Number and size of sequenced-clone contigs, sequenced-clone-contig scaffolds and those ®ngerprint clone contigs (see Box 1) that contain sequenced clones; some small ®ngerprint clone contigs do notas yet have associated sequence. UL, ®ngerprint clone contigs that could not reliably be placed on a chromosome. These length estimates are from the draft genome sequence, in which gaps betweensequence contigs are arbitrarily represented with 100 Ns and gaps between sequence clone contigs with 50,000 Ns for `bridged gaps' and 100,000 Ns for `unbridged gaps'. These arbitrary values differminimally from empirical estimates of gap size (see text), and using the empirically derived estimates would change the N50 lengths presented here only slightly. For un®nished chromosomes, the N50 lengthranges from 1.5 to 3 times the arithmetic mean for sequenced-clone contigs, 1.5 to 3 times for sequenced-clone-contig scaffolds, and 1.5 to 6 times for ®ngerprint clone contigs with sequence.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd871