Initial sequencing and analysis of the human genome - Vitagenes

articles®ngerprint map. However, many involve STSs that have beenlocalized on only one or two of the previous maps or that occuras isolated discrepancies in con¯ict with several ¯anking STSs.Many of these cases are probably due to errors in the previousmaps (with error rates for individual maps estimated at 1±2% 100 ).Others may be due to incorrect assignment of the STSs to the draftgenome sequence (by the electronic polymerase chain reaction(e-PCR) computer program) or to database entries that containsequence data from more than one clone (owing to crosscontamination).Graphical views of the independent data sets were particularlyuseful in detecting problems with order or orientation (Fig. 5).Areas of con¯ict were reviewed and corrected if supported by theunderlying data. In the version discussed here, there were 41sequenced clones falling in 14 sequenced-clone contigs with STScontent information from multiple maps that disagreed with the¯anking clones or sequenced-clone contigs; the placement of theseclones thus remains suspect. Four of these instances suggest errorsin the ®ngerprint map, whereas the others suggest errors in thelayout of sequenced clones. These cases are being investigated andwill be corrected in future versions.Assembly of the sequenced clones. We assessed the accuracy of theassembly by using a set of 148 draft clones comprising 22.4 Mb forwhich ®nished sequence subsequently became available 104 . Theinitial sequence contigs lack information about order and orientation,and GigAssembler attempts to use linking data to infer suchinformation as far as possible 104 . Starting with initial sequencecontigs that were unordered and unoriented, the program placed90% of the initial sequence contigs in the correct orientation and85% in the correct order with respect to one another. In a separatetest, GigAssembler was tested on simulated draft data producedfrom ®nished sequence on chromosome 22 and similar results wereobtained.Some problems remain at all levels. First, errors in the initialsequence contigs persist in the merged sequence contigs built fromthem and can cause dif®culties in the assembly of the draft genomesequence. Second, GigAssembler may fail to merge some overlappingsequences because of poor data quality, allelic differences ormisassemblies of the initial sequence contigs; this may result inapparent local duplication of a sequence. We have estimated byvarious methods the amount of such artefactual duplication in theassembly from these and other sources to be about 100 Mb. On theother hand, nearby duplicated sequences may occasionally be incorrectlymerged. Some sequenced clones remain incorrectly placed onthe layout, as discussed above, and others (, 0.5%) remain unplaced.The ®ngerprint map has undoubtedly failed to resolve some closelyrelated duplicated regions, such as the Williams region and severalhighly repetitive subtelomeric and pericentric regions (see below).Detailed examination and sequence ®nishing may be required tosort out these regions precisely, as has been done with chromosomeY 89 . Finally, small sequenced-clone contigs with limited or no STSTable 9 Distribution of PHRAP scores in the draft genome sequencePHRAP scorePercentage of bases in the draftgenome sequence0±9 0.610±19 1.320±29 2.230±39 4.840±49 8.150±59 8.760±69 9.070±79 12.180±89 17.3.90 35.9.............................................................................................................................................................................PHRAP scores are a logarithmically based representation of the error probability. A PHRAP score ofX corresponds to an error probability of 10 -X/10 . Thus, PHRAP scores of 20, 30 and 40 correspond toaccuracy of 99%, 99.9% and 99.99%, respectively. PHRAP scores are derived from qualityscores of the underlying sequence reads used in sequence assembly. See http://www.genome.washington.edu/UWGC/analysistools/phrap.htm.landmark content remain dif®cult to place. Full utilization ofthe higher resolution radiation hybrid map (the TNG map) mayhelp in this 95 . Future targeted FISH experiments and increased mapcontinuity will also facilitate positioning of these sequences.Genome coverageWe next assessed the nature of the gaps within the draft genomesequence, and attempted to estimate the fraction of the humangenome not represented within the current version.Gaps in draft genome sequence coverage. There are three types ofgap in the draft genome sequence: gaps within un®nishedsequenced clones; gaps between sequenced-clone contigs, butwithin ®ngerprint clone contigs; and gaps between ®ngerprintclone contigs. The ®rst two types are relatively straightforward toclose simply by performing additional sequencing and ®nishing onalready identi®ed clones. Closing the third type may require screeningof additional large-insert clone libraries and possibly newtechnologies for the most recalcitrant regions. We consider thesethree cases in turn.We estimated the size of gaps within draft clones by studyinginstances in which there was substantial overlap between a draftclone and a ®nished clone, as described above. The average gap sizein these draft sequenced clones was 554 bp, although the preciseestimate was sensitive to certain assumptions in the analysis.Assuming that the sequence gaps in the draft genome sequenceare fairly represented by this sample, about 80 Mb or about 3%(likely range 2±4%) of sequence may lie in the 145,514 gaps withindraft sequenced clones.The gaps between sequenced-clone contigs but within ®ngerprintclone contigs are more dif®cult to evaluate directly, because thedraft genome sequence ¯anking many of the gaps is often notprecisely aligned with the ®ngerprinted clones. However, most aremuch smaller than a single BAC. In fact, nearly three-quarters ofthese gaps are bridged by one or more individual BACs, as indicatedby linking information from BAC end sequences. We measured thesizes of a subset of gaps directly by examining restriction fragment®ngerprints of overlapping clones. A study of 157 `bridged' gaps and55 `unbridged' gaps gave an average gap size of 25 kb. Allowing for thepossibility that these gaps may not be fully representative and thatsome restriction fragments are not included in the calculation, a moreconservative estimate of gap size would be 35 kb. This would indicatethat about 150 Mb or 5% of the human genome may reside in the4,076 gaps between sequenced-clone contigs. This sequence shouldbe readily obtained as the clones spanning them are sequenced.The size of the gaps between ®ngerprint clone contigs wasestimated by comparing the ®ngerprint maps to the essentiallycompleted chromosomes 21 and 22. The analysis shows that the®ngerprinted BAC clones in the global database cover 97±98% ofthe sequenced portions of those chromosomes 86 . The publishedsequences of these chromosomes also contain a few small gaps (5and 11, respectively) amounting to some 1.6% of the euchromaticsequence, and do not include the heterochromatic portion. Thissuggests that the gaps between contigs in the ®ngerprint mapcontain about 4% of the euchromatic genome. Experience withclosure of such gaps on chromosomes 20 and 7 suggests that manyof these gaps are less than one clone in length and will be closed byclones from other libraries. However, recovery of sequence fromthese gaps represents the most challenging aspect of producing acomplete ®nished sequence of the human genome.As another measure of the representation of the BAC libraries,Riethman 109 has found BAC or cosmid clones that link to telomerichalf-YACs or to the telomeric sequence itself for 40 of the 41 nonsatellitetelomeres. Thus, the ®ngerprint map appears to have nosubstantial gaps in these regions. Many of the pericentric regions arealso represented, but analysis is less complete here (see below).Representation of random raw sequences. In another approach tomeasuring coverage, we compared a collection of random rawsequence reads to the existing draft genome sequence. In principle,874 © 2001 Macmillan Magazines Ltd NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com