Initial sequencing and analysis of the human genome - Vitagenes

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articlesThe pericentromeric regions are structurally very complex, asillustrated for chromosome 21 in Fig. 32a. The pericentromericregions appear to have been bombarded by successive insertions ofduplications; the insertion events must be fairly recent because thedegree of sequence conservation with the genomic source loci isfairly high (90±100%, with an apparent peak around 96%). Distinctinsertions are typically separated by AT-rich or GC-rich minisatellite-likerepeats that have been hypothesized to have a functionalrole in targeting duplications to these regions 233,241 .A single genomic source locus often gives rise to pericentromericcopies on multiple chromosomes, with each having essentially thesame breakpoints and the same degree of divergence. An example ofsuch a source locus on Xq28 is shown in Fig. 32b. Phylogeneticanalysis has suggested a two-step mechanism for the origin anddispersal of these segments, whereby an initial segmental duplicationin the pericentromeric region of one chromosome occurs and isthen redistributed as part of a larger cassette to other such regions 242 .A comprehensive analysis for all chromosomes will have to awaitcomplete sequencing of the genome, but the evidence from the draftgenome sequence indicates that the same picture is likely to be seenthroughout the genome. Several papers have analysed ®nishedsegments within pericentromeric regions of chromosomes 2(160 kb), 10 (400 kb) and 16 (300 kb), all of which show extensiveinterchromosomal segmental duplication 215,219,232,233 . An examplefrom another pericentromeric region on chromosome 11 isshown in Fig. 32c. Interchromosomal duplications in subtelomericregions also appear to be a fairly general phenomenon, as illustratedby a large tract (,500 kb) of complex duplication on chromosome 7(Fig. 32d).The explanation for the clustering of segmental duplications maybe that the genome has a damage-control mechanism wherebychromosomal breakage products are preferentially inserted intopericentromeric and, to a lesser extent, subtelomeric regions. Thepossibility of a speci®c mechanism for the insertion of thesesequences has been suggested on the basis of the unusual sequencesfound ¯anking the insertions. Although it is also possible that theseregions simply have greater tolerance for large insertions, manylarge gene-poor `deserts' have been identi®ed 93 and there is noaccumulation of duplicated segments within these regions. Alongwith the fact that transitions between duplicons (from differentregions of the genome) occur at speci®c sequences, this suggests thatactive recruitment of duplications to such regions may occur. In anycase, the duplicated regions are in general young (with manyduplications showing ,6% nucleotide divergence from theirsource loci) and in constant ¯ux, both through additional duplicationsand by large-scale exchange among similar chromosomalenvironments. There is evidence of structural polymorphism inthe human population, such as the presence or absence of olfactoryreceptor segments located within the telomeric regions of severalhuman chromosomes 226,227 .Genome-wide analysis of segmental duplications. We also performeda global genome-wide analysis to characterize the amountof segmental duplication in the genome. We `repeat-masked' theknown interspersed repeats in the draft genome sequence andcompared the remaining draft genomic sequence with itself in amassive all-by-all BLASTN similarity search. We excluded matchesin which the sequence identity was so high that it might re¯ectartefactual duplications resulting from a failure to overlap sequencecontigs correctly in assembling the draft genome sequence. Speci-®cally, we considered only matches with less than 99.5% identity for®nished sequence and less than 98% identity for un®nishedsequence.We took several approaches to avoid counting artefactual duplicationsin the sequence. In the ®rst approach, we studied only®nished sequence. We compared the ®nished sequence with itself,to identify segments of at least 1 kb and 90±99.5% sequenceidentity. This analysis will underestimate the extent of segmentalduplication, because it requires that at least two copies of thesegment are present in the ®nished sequence and because sometrue duplications have over 99.5% identity.The ®nished sequence consists of at least 3.3% segmental duplication(Table 16). Interchromosomal duplication accounts forabout 1.5% and intrachromosomal duplication for about 2%,with some overlap (0.2%) between these categories. We analysedthe lengths and divergence of the segmental duplications (Fig. 33).The duplications tend to be large (10±50 kb) and highly homologous,especially for the interchromosomal segments. Thesequence divergence for the interchromosomal duplicationsappears to peak between 96.5% and 97.5%. This may indicatethat interchromosomal duplications occurred in a punctuatedmanner. It will be intriguing to investigate whether such genomicupheaval has a role in speciation events.In a second approach, we compared the entire human draftgenome sequence (®nished and un®nished) with itself to identifyduplications with 90±98% sequence identity (Table 17). The draftgenome sequence contains at least 3.6% segmental duplication. Theactual proportion will be signi®cantly higher, because we excludedmany true matches with more than 98% sequence identity (at least1.1% of the ®nished sequence). Although exact measurement mustawait a ®nished sequence, the human genome seems likely tocontain about 5% segmental duplication, with most of thissequence in large blocks (. 10 kb). Such a high proportion oflarge duplications clearly distinguishes the human genome fromother sequenced genomes, such as the ¯y and worm (Table 18).The structure of large highly paralogous regions presents one ofthe `serious and unanticipated challenges' to producing a ®nishedsequence of the genome 46 . The absence of unique STS or ®ngerprintsignatures over large genomic distances (,1 Mb) and the highdegree of sequence similarity makes the distinction between paralogoussequence variation and allelic polymorphism problematic.Furthermore, the fact that such regions frequently harbour intron±exon structures of genuine unique sequence will complicate effortsto generate a genome-wide SNP map. The data indicate that amodest portion of the human genome may be relatively recalcitrantto genomic-based methods for SNP detection. Owing to theirrepetitive nature and their location in the genome, segmentalTable 16 Fraction of ®nished sequence in inter- and intrachromosomalduplicationsChromosome Intrachromosomal (%) Interchromosomal (%) All (%)1 1.4 0.5 1.92 0.1 0.6 0.73 0.3 1.1 1.14 0.0 1.0 1.05 0.6 0.3 0.96 0.8 0.4 1.17 3.4 1.3 4.18 0.3 0.1 0.39 0.8 2.9 3.710 2.1 0.8 2.911 1.2 2.1 2.312 1.5 0.3 1.813 0.0 0.5 0.514 0.6 0.4 1.015 3.0 6.9 6.916 4.5 2.0 5.817 1.6 0.3 1.818 0.0 0.7 0.719 3.6 0.3 3.820 0.2 0.3 0.521 1.4 1.6 3.022 6.1 2.6 7.5X 1.8 3.2 5.0Y 12.1 16.0 27.4Un 0.0 0.5 0.5Total 2.0 1.5 3.3.............................................................................................................................................................................Excludes duplications with identities .99.5% to avoid artefactual duplication due to incompletemerger in the assembly process. Calculation was performed on the ®nished sequence available inSeptember 2000 and re¯ects the duplications found within the total amount of ®nished sequencethen. Note that there is some overlap between the interchromosomal and intrachromosomal sets.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd891
articlesaSum of aligned bases (kb)c1,200,000Sum of aligned bases (kb)900,000800,000700,000600,000500,000400,000300,000200,000100,00001,000,000800,000600,000400,000200,0000Intrachromosomal90 90.5 91 91.5 92 92.5 93 93.5 94 94.5 95 95.5 96 96.5 97 97.5 98 98.5 99Similarity (%)bSum of aligned bases (kb)dSum of aligned bases (kb)1,600,0001,400,0001,200,0001,000,000800,000600,000400,000200,00004,000,0003,500,0003,000,0002,500,0002,000,0001,500,0001,000,000500,000Interchromosomal90 90.5 91 91.5 92 92.5 93 93.5 94 94.5 95 95.5 96 96.5 97 97.5 98 98.5 99Similarity (%)01 2 3 4 5 6 7 8 9 10–19 20–29 30–39 40–49 50+ 1 2 3 4 5 6 7 8 9 10–19 20–29 30–39 40–49 50+Length of alignment (kb)Length of alignment (kb)Figure 33 a±d, Sequence properties of segmental duplications. Distributions of lengthand per cent nucleotide identity for segmental duplications are shown as a function of thenumber of aligned bp, for the subset of ®nished genome sequence. Intrachromosomal,red; interchromosomal, blue.duplications may well be underestimated by the current analysis. Anunderstanding of the biology, pathology and evolution of theseduplications will require specialized efforts within these exceptionalregions of the human genome. The presence and distribution ofsuch segments may provide evolutionary fodder for processes ofexon shuf¯ing and a general increase in protein diversity associatedwith domain accretion. It will be important to consider bothgenome-wide duplication events and more restricted punctuatedevents of genome duplication as forces in the evolution of vertebrategenomes.Table 17 Fraction of the draft genome sequence in inter- and intrachromosomalduplicationsChromosome Intrachromosomal (%) Interchromosomal (%) All (%)1 2.1 1.7 3.42 1.6 1.6 2.63 1.8 1.4 2.74 1.5 2.2 3.05 1.0 0.9 1.86 1.5 1.4 2.77 3.6 1.8 4.58 1.2 1.5 2.19 2.1 2.3 3.810 3.3 2.0 4.711 2.7 1.4 3.712 2.1 1.2 2.813 1.7 1.6 3.014 0.6 0.6 1.215 4.1 4.4 6.716 3.4 3.4 5.517 4.4 1.7 5.718 0.9 1.0 1.919 5.4 1.6 6.320 0.8 1.4 2.021 1.9 4.0 4.822 6.8 7.7 11.9X 1.2 1.1 2.2Y 10.9 13.1 20.8NA 2.3 7.8 8.3UL 11.6 20.8 22.2Total 2.3 2.0 3.6.............................................................................................................................................................................Excludes duplications with identities .98% to avoid artefactual duplication due to incompletemerger in the assembly process. Calculation was performed on an earlier version of the draftgenome sequence based on data available in July 2000 and re¯ects the duplications found withinthe total amount of ®nished sequence then. Note that there is some overlap between theinterchromosomal and intrachromosomal sets.Gene content of the human genomeGenes (or at least their coding regions) comprise only a tiny fractionof human DNA, but they represent the major biological function ofthe genome and the main focus of interest by biologists. They arealso the most challenging feature to identify in the human genomesequence.The ultimate goal is to compile a complete list of all human genesand their encoded proteins, to serve as a `periodic table' forbiomedical research 243 . But this is a dif®cult task. In organismswith small genomes, it is straightforward to identify most genes bythe presence of long ORFs. In contrast, human genes tend to havesmall exons (encoding an average of only 50 codons) separated bylong introns (some exceeding 10 kb). This creates a signal-to-noiseproblem, with the result that computer programs for direct geneprediction have only limited accuracy. Instead, computationalprediction of human genes must rely largely on the availability ofcDNA sequences or on sequence conservation with genes andproteins from other organisms. This approach is adequate forstrongly conserved genes (such as histones or ubiquitin), but maybe less sensitive to rapidly evolving genes (including many crucial tospeciation, sex determination and fertilization).Here we describe our efforts to recognize both the RNA genes andprotein-coding genes in the human genome. We also study theproperties of the predicted human protein set, attempting to discernhow the human proteome differs from those of invertebrates such asworm and ¯y.Noncoding RNAsAlthough biologists often speak of a tight coupling between `genesTable 18 Cross-species comparison for large, highly homologous segmentalduplicationsPercentage of genome (%)Fly Worm Human (®nished)*. 1 kb 1.2 4.25 3.25. 5 kb 0.37 1.50 2.86. 10 kb 0.08 0.66 2.52.............................................................................................................................................................................* This is an underestimate of the total amount of segmental duplication in the human genomebecause it only re¯ects duplication detectable with available ®nished sequence. The proportion ofsegmental duplications of . 1 kb is probably about 5% (see text).892 © 2001 Macmillan Magazines Ltd NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com
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Initial sequencing and analysis of the human genome - Vitagenes

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