Initial sequencing and analysis of the human genome - Vitagenes

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articlesTable 14 SSR content of the human genomeLength of repeat unit Average bases per Mb Average number of SSRelements per Mb1 1,660 36.72 5,046 43.13 1,013 11.84 3,383 32.55 2,686 17.66 1,376 15.27 906 8.48 1,139 11.19 900 8.610 1,576 8.611 770 8.7.............................................................................................................................................................................SSRs were identi®ed by using the computer program Tandem Repeat Finder with the followingparameters: match score 2, mismatch score 3, indel 5, minimum alignment 50, maximum repeatlength 500, minimum repeat length 1.tellites, whereas those with longer repeat units (n = 14±500 bases)are often termed minisatellites. With the exception of poly(A) tailsfrom reverse transcribed messages, SSRs are thought to arise byslippage during DNA replication 212,213 .We compiled a catalogue of all SSRs over a given length in thehuman draft genome sequence, and studied their properties(Table 14). SSRs comprise about 3% of the human genome, withthe greatest single contribution coming from dinucleotide repeats(0.5%). (The precise criteria for the number of repeat units and theextent of divergence allowed in an SSR affect the exact census, butnot the qualitative conclusions.)There is approximately one SSR per 2 kb (the number of nonoverlappingtandem repeats is 437 per Mb). The catalogue con®rmsvarious properties of SSRs that have been inferred from samplingapproaches (Table 15). The most frequent dinucleotide repeats areAC and AT (50 and 35% of dinucleotide repeats, respectively),whereas AG repeats (15%) are less frequent and GC repeats (0.1%)are greatly under-represented. The most frequent trinucleotides areAAT and AAC (33% and 21%, respectively), whereas ACC (4.0%),AGC (2.2%), ACT (1.4%) and ACG (0.1%) are relatively rare.Overall, trinucleotide SSRs are much less frequent than dinucleotideSSRs 214 .SSRs have been extremely important in human genetic studies,because they show a high degree of length polymorphism in thehuman population owing to frequent slippage by DNA polymeraseduring replication. Genetic markers based on SSRsÐparticularly(CA) n repeatsÐhave been the workhorse of most human diseasemappingstudies 101,102 . The availability of a comprehensive catalogueof SSRs is thus a boon for human genetic studies.The SSR catalogue also allowed us to resolve a mystery regardingmammalian genetic maps. Such genetic maps in rat, mouse andhuman have a de®cit of polymorphic (CA) n repeats on chromosomeX 30,101 . There are two possible explanations for this de®cit. Theremay simply be fewer (CA) n repeats on chromosome X; or (CA) nrepeats may be as dense on chromosome X but less polymorphic inthe population. In fact, analysis of the draft genome sequence showsthat chromosome X has the same density of (CA) n repeats per Mb asthe autosomes (data not shown). Thus, the de®cit of polymorphicmarkers relative to autosomes results from population geneticforces. Possible explanations include that chromosome X has asmaller effective population size, experiences more frequent selectivesweeps reducing diversity (owing to its hemizygosity in males),or has a lower mutation rate (owing to its more frequent passagethrough the less mutagenic female germline). The availability of thedraft genome sequence should provide ways to test these alternativeexplanations.Segmental duplicationsA remarkable feature of the human genome is the segmentalduplication of portions of genomic sequence 215±217 . Such duplicationsinvolve the transfer of 1±200-kb blocks of genomic sequenceto one or more locations in the genome. The locations of bothdonor and recipient regions of the genome are often not tandemlyarranged, suggesting mechanisms other than unequal crossing-overfor their origin. They are relatively recent, inasmuch as strongsequence identity is seen in both exons and introns (in contrast toregions that are considered to show evidence of ancient duplications,characterized by similarities only in coding regions). Indeed,many such duplications appear to have arisen in very recentevolutionary time, as judged by high sequence identity and bytheir absence in closely related species.Segmental duplications can be divided into two categories. First,interchromosomal duplications are de®ned as segments that areduplicated among nonhomologous chromosomes. For example, a9.5-kb genomic segment of the adrenoleukodystrophy locus fromXq28 has been duplicated to regions near the centromeres ofchromosomes 2, 10, 16 and 22 (refs 218, 219). Anecdotal observationssuggest that many interchromosomal duplications map near thecentromeric and telomeric regions of human chromosomes 218±233 .The second category is intrachromosomal duplications, whichoccur within a particular chromosome or chromosomal arm. Thiscategory includes several duplicated segments, also known as lowcopy repeat sequences, that mediate recurrent chromosomal structuralrearrangements associated with genetic disease 215,217 . Exampleson chromosome 17 include three copies of a roughly 200-kb repeatseparated by around 5 Mb and two copies of a roughly 24-kb repeatseparated by 1.5 Mb. The copies are so similar (99% identity) thatparalogous recombination events can occur, giving rise to contiguousgene syndromes: Smith±Magenis syndrome and Charcot±Marie±Tooth syndrome 1A, respectively 34,234 . Several other examplesare known and are also suspected to be responsible for recurrentmicrodeletion syndromes (for example, Prader±Willi/Angelman,Table 15 SSRs by repeat unitRepeat unitNumber of SSRs per MbAC 27.7AT 19.4AG 8.2GC 0.1AAT 4.1AAC 2.6AGG 1.5AAG 1.4ATG 0.7CGG 0.6ACC 0.4AGC 0.3ACT 0.2ACG 0.0.............................................................................................................................................................................SSRs were identi®ed as in Table 14.Figure 30 Duplication landscape of chromosome 22. The size and location ofintrachromosomal (blue) and interchromosomal (red) duplications are depicted forchromosome 22q, using the PARASIGHT computer program (Bailey and Eichler,unpublished). Each horizontal line represents 1 Mb (ticks, 100-kb intervals). Thechromosome sequence is oriented from centromere (top left) to telomere (bottom right).Pairwise alignments with . 90% nucleotide identity and . 1 kb long are shown. Gapswithin the chromosomal sequence are of known size and shown as empty space.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd889
articlesvelocardiofacial/DiGeorge and Williams' syndromes 215,235±240 ).Until now, the identi®cation and characterization of segmentalduplications have been based on anecdotal reportsÐfor example,®nding that certain probes hybridize to multiple chromosomal sitesor noticing duplicated sequence at certain recurrent chromosomalbreakpoints. The availability of the entire genomic sequence willmake it possible to explore the nature of segmental duplicationsmore systematically. This analysis can begin with the current state ofthe draft genome sequence, although caution is required becausesome apparent duplications may arise from a failure to mergesequence contigs from overlapping clones. Alternatively, erroneousFigure 31 Duplication landscape of chromosome 21. The size and location ofintrachromosomal (blue) and interchromosomal (red) duplications are depicted along thesequence of the long arm of chromosome 21. Gaps between ®nished sequence aredenoted by empty space but do not represent actual gap size.assembly of closely related sequences from nonoverlapping clonesmay underestimate the true frequency of such features, particularlyamong those segments with the highest sequence similarity. Accordingly,we adopted a conservative approach for estimating suchduplication from the available draft genome sequence.Pericentromeres and subtelomeres. We began by re-evaluating the®nished sequences of chromosomes 21 and 22. The initial papers onthese chromosomes 93,94 noted some instances of interchromosomalduplication near each centromere. With the ability now to comparethese chromosomes to the vast majority of the genome, it isapparent that the regions near the centromeres consist almostentirely of interchromosomal duplicated segments, with littleor no unique sequence. Smaller regions of interchromosomalduplication are also observed near the telomeres.Chromosome 22 contains a region of 1.5 Mb adjacent to thecentromere in which 90% of sequence can now be recognized toconsist of interchromosomal duplication (Fig. 30). Conversely, 52%of the interchromosomal duplications on chromosome 22 werelocated in this region, which comprises only 5% of the chromosome.Also, the subtelomeric end consists of a 50-kb region consistingalmost entirely of interchromosomal duplications.Chromosome 21 presents a similar landscape (Fig. 31). The ®rst1 Mb after the centromere is composed of interchromosomalrepeats, as well as the largest (. 200 kb) block of intrachromosomallyduplicated material. Again, most interchromosomal duplicationson the chromosome map to this region and the mostsubtelomeric region (30 kb) shows extensive duplication amongnonhomologous chromosomes.aSequence identity (%)10099989796959493929190100 200 300 400 500cSequence identity (%)10099989796959493929190100 200 300 400 500Sequence identity (%)10099989796959493929190600 700 800 900 1,000Sequence identity (%)10099989796959493929190600 700 800 900 1,000Sequence identity (%)100999897969594939291901,100 1,200 1,300 1,400 1,500Sequence identity (%)100999897969594939291901,100 1,200 1,300 1,400 1,500bSequence identity10099989796959493929190CTR CMD ALD320 340 360 380 420400 440dSequence identity (%)Sequence identity (%)1009998979695949392919010099989796959493929190100 200 300 400 500600Figure 32 Mosaic patterns of duplications. Panels depict various patterns of duplicationwithin the human genome (PARASIGHT). For each region, a segment of draft genomesequence (100±500 kb) is shown with both interchromosomal (red) and intrachromosomal(blue) duplications displayed along the horizontal line. Below the line, each separatesequence duplication is indicated (with a distinct colour) relative to per cent nucleotideidentity for the duplicated segment (y axis). Black bars show the relative locations of largeblocks of heterochromatic sequences (alpha, gamma and HSAT sequence). a, An activepericentromeric region on chromosome 21. b, An ancestral region from Xq28 that hascontributed various `genic' segments to pericentromeric regions. c, A pericentromeric890 © 2001 Macmillan Magazines Ltd NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com
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