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Integration of cytogenetic landmarks into the draft sequence of the human genome

Integration of cytogenetic landmarks into the draft sequence of the human genome letters to nature 11. Mohrenweiser, H. W., Tsujimoto, S., Gordon, L. & Olsen, A. S. Regions of sex-specific hypo- and low recombination. Nucleotide and haplotype diversity will also hyper-recombination identified through integration of 180 genetic markers into the metric physical probably parallel recombination rates. Although our baseline long- map of human chromosome 19. Genomics 47, 153 – 162 (1998). range recombination rates will be useful, they should be recalculated 12. Nicolas, A. Relationship between transcription and initiation of meiotic recombination: toward when the human genomic sequences are finished and as higher chromatin accessibility. Proc. Natl Acad. Sci. USA 95, 87– 89 (1998). 13. Wahls, W. P. Meiotic recombination hotspots: shaping the genome and insights into hypervariable resolution genetic maps become available. In the more distant minisatellite DNA change. Curr. Top. Dev. Biol. 37, 37 – 75, (1998). future, genotyping greater numbers of reference families at much 14. Faris, J. D., Haen, K. M. & Gill, B. S. Saturation mapping of a gene-rich recombination hot spot region higher polymorphism densities will lead to short-range maps of in wheat. Genetics 154, 823 – 835 (2000). 15. Kliman, R. M. & Hey, J. Reduced natural selection associated with low recombination in Drosophila recombination hot spots. M melanogaster. Mol. Biol. Evol. 10, 1239– 1258 (1993). 16. Chen, T. L. & Manuelidis, L. SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size. Methods Chromosoma 98, 309 – 316 (1989). 17. Payseur, B. A. & Nachman, M. W. Microsatellite variation and recombination rate in the human Connection of genetic and physical maps genome. Genetics 156, 1285– 1298 (2000). We used short, single-pass genomic sequences and/or PCR primer sequences for STRPs to 18. Nachman, M. W., Bauer, V. L., Crowell, S. L. & Aquadro, C. F. DNA variability and recombination rates identify draft or finished bacterial artificial chromosome (BAC) or cosmid sequences at X-linked loci in humans. Genetics 150, 1133– 1141 (1998). 27 28 within GenBank that encompass the STRPs using BLAST and ePCR . Blast criteria were 19. Nachman, M. W. & Crowell, S. L. Contrasting evolutionary histories of two introns of the Duchenne −50 score (bits) . 200, expect (E) value , e , and ratio of matched bases to marker sequence muscular dystrophy gene, Dmd, in humans. Genetics 155, 1855 – 1864 (2000). length . 85%. ePCR criteria were no more than one base mismatch in each primer and 20. Majewski, J. & Ott, J. GT repeats are associated with recombination on human chromosome 22. size of PCR product within allele size range for the STRP. About 75% of the STRPs were Genome Res. 10, 1108– 1114 (2000). connected to the long genomic sequences. The reasons for failure of the remaining 25% are 21. Bernardi, G. Isochores and the evolutionary genomics of vertebrates. Gene 241, 3 – 17 (2000). not fully understood, but include absence of the corresponding sequence in GenBank and 22. Eisenbarth, I., Vogel, G., Krone, W., Vogel, W. & Assum, G. An isochore transition in the NF1 gene poor quality of the STRP sequences. As the genetic maps are marker rich, the absence of region coincides with a switch in the extent of linkage disequilibrium. Am. J. Hum. Genet. 67, 873 – 880 25% was not a serious limitation. Tables of STRPs with GenBank sequence accession (2000). numbers for encompassing BACs, genetic map positions and recombination rates are 23. Yu, J. et al. Individual variation in recombination among human males. Am. J. Hum. Genet. 59, 1186– available from the Marshfield web site. 1192 (1996). 24. Lien, S., Szyda, J., Schechinger, B., Rappold, G. & Arnheim, N. Evidence for heterogeneity in recombination in the human pseudoautosomal region: High resolution analysis by sperm typing and Determination of recombination rates radiation-hybrid mapping. Am. J. Hum. Genet. 66, 557 – 566 (2000). For each sequence assembly we built new female, male and sex-average genetic maps, using 25. Carrington, M. Recombination within the human MHC. Immunol Rev. 167, 245– 256 (1999). the marker order provided by the assemblies and using the genotyping data from the eight 26. Jeffreys, A. J., Ritchie, A. & Neumann, R. High resolution analysis of haplotype diversity and meiotic CEPH reference families . We fitted cubic splines to plots of genetic versus physical crossover in the human TAP2 recombination hot spot. Hum. Mol. Genet. 9, 725 – 733 (2000). distance, and from these curves we obtained recombination rates as first derivatives . The 27. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search statistical significance of the recombination rates was estimated by computer simulation of programs. Nucleic Acids Res. 25, 3389 (1997). 1,000 iterations of recombination within each interval between markers, assuming a 28. Schuler, G. D. Sequence mapping by electronic PCR. Genome Res. 7, 541 – 550 (1997). constant level of recombination across the genome for each sex. The constant levels of 29. Zhao, C., Heil, J., & Weber, J. L. A genome wide portrait of short tandem repeats. Am. J. Hum. Genet. recombination were taken as the total genetic lengths of all the assemblies analysed divided 65, (Suppl.) A102 (1999). by the total physical lengths of these assemblies. 30. Huttley, G., A., Smith, M. W., Carrington, M. & O’Brien, S. J. A scan for linkage disequilibrium across the human genome. Genetics 152, 1711– 1722 (1999). Computation of marker and sequence parameters Supplementary information is available from Nature’s World-Wide Web site We calculated STRP heterozygosities using genotypes of individuals within the eight (http://www.nature.com) or as paper copy from the London editorial office of Nature, and CEPH families. We obtained STRP positions relative to centromeres and telomeres as the from the Marshfield Web site (http://research.marshfieldclinic.org/genetics). fractional sex-average genetic map distances from the centromeres to the telomeres (value of 0 for a STRP at the centromere and 1.0 for a STRP at the telomere) . GC content and Acknowledgements STR densities were obtained from programs written and tested at Marshfield . STR densities were measured as numbers of runs of non-interrupted repeats rather than total This work was supported by contracts from the US National Institutes of Health and numbers of repeats. Minimum values of n for (A) ,(AC) ,(AGAT) , (AAN) and n n n n Department of Energy. Assistance was also provided by the chromosome 6 and 20 project (AAAN) sequences were 12, 11 or 19 ((AC) ), 5, 7 and 5, respectively. We obtained n n groups at the Sanger Centre, supported by the Wellcome Trust. interspersed repetitive element densities using the program Repeat Masker Correspondence and requests for materials should be addressed to J.L.W. (http://ftp.genome.washington.edu/RM/RepeatMasker.html). SINEs and LINEs were (e-mail: [email protected]fldclin.edu). defined by Repeat Masker and consist primarily of Alu and L1 elements, respectively. We computed all DNA sequence parameters over 250-kb windows centred about each STRP. For markers # 125 kb from the ends of the sequence assemblies, we defined the window as the 125 kb of proximal sequence plus all available distal sequence. Unknown bases in the sequence assemblies were excluded from analysis. All parameters were corrected for reduced window size owing to unknown bases or proximity to ends. ................................................................. Measurement of linkage disequilibrium Integration of cytogenetic landmarks Recombination deserts and jungles were selected as those chromosomal regions with sex- average recombination rates of ,0.3 or .3.0, respectively. We measured linkage disequili- into the draft sequence of the human brium for all pairs of STRPs within the deserts (449 pairs) and jungles (467 pairs) using Fisher’s exact test . Only disequilibrium results that were significant at P # 0.01 were plotted genome in Fig. 2. An overall P-value was obtained by a permutation test treating the regions as units in order to account for the dependence between marker pairs within a region. The BAC Resource Consortium* Received 27 October; accepted 8 December 2000. 1. The BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the * Authorship of this paper should be cited using the names of authors that appear at human genome. Nature 409, 953 – 958 (2001). the end. 2. The International Human Genome Mapping Consortium. A physical map of the human genome. Nature 409, 934 – 941 (2001). ................. ......................... ......................... ......................... ......................... ......................... 3. Deloukas, P. et al. A physical map of 30,000 human genes. Science 282, 744 – 746 (1998). We have placed 7,600 cytogenetically defined landmarks on the 4. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human draft sequence of the human genome to help with the character- genome. Nature 409, 860 – 921 (2001). 5. Broman, K. W., Murray, J. C., Sheffield, V. C., White, R. L. & Weber, J. L. Comprehensive human genetic ization of genes altered by gross chromosomal aberrations that maps: Individual and sex-specific variation in recombination. Am. J. Hum. Genet. 63, 861–869 (1998). cause human disease. The landmarks are large-insert clones 6. Dunham, I. et al. The DNA sequence of human chromosome 22. Nature 402, 489 – 495 (1999). mapped to chromosome bands by fluorescence in situ hybridiza- 7. Hattori, M. et al. The DNA sequence of human chromosome 21. Nature 405, 311 – 319 (2000). tion. Each clone contains a sequence tag that is positioned on the 8. Fain, P. R., Kort, E. N., Yousry, C., James, M. R. & Litt, M. A high resolution CEPH crossover mapping panel and integrated map of chromosome 11. Hum. Mol. Genet. 5, 1631 – 1636 (1996). genomic sequence. This genome-wide set of sequence-anchored 9. Bouffard, G. G. et al. A physical map of human chromosome 7: An integrated YAC contig map with clones allows structural and functional analyses of the genome. average STS spacing of 79 kb. Genome Res. 7, 673 – 692 (1997). This resource represents the first comprehensive integration of 10. Nagaraja, R. et al. X chromosome map at 75-kb STS resolution, revealing extremes of recombination cytogenetic, radiation hybrid, linkage and sequence maps of the and GC content. Genome Res. 7, 210 – 222 (1997). NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 953 © 2001 Macmillan Magazines Ltd letters to nature human genome; provides an independent validation of the The set, which consists primarily of bacterial artificial chromosome 1,2 sequence map and framework for contig order and orientation; (BAC) clones, includes clones targeted to contain sequence-tagged surveys the genome for large-scale duplications, which are likely sites (STSs) ordered along the genome by genetic linkage or radiation to require special attention during sequence assembly; and allows hybrid mapping (for well ordered and distributed coverage); clones a stringent assessment of sequence differences between the dark randomly selected for end sequencing from the RPCI-11 library (for and light bands of chromosomes. It also provides insight into coverage of regions low in STSs); clones identified during intense large-scale chromatin structure and the evolution of chromo- mapping efforts that preceded sequencing of some chromosomes somes and gene families and will accelerate our understanding of (for denser coverage); and clones suspected of being partially the molecular bases of human disease and cancer. With the draft of the human genome available , scientists can conduct global analyses of its gene content, structure, function and ab variation. One important challenge is to define the genetic con- tribution to human diseases. For many developmental disorders, inherited conditions and cancers, gross chromosomal aberrations provide clues to the locations of the causative molecular defects. nl19 These aberrations are visible as alterations in chromosomal banding der(11) patterns or in the number or relative positions of DNA sequences der(19) labelled by fluorescence in situ hybridization (FISH) . Although tracing gross abnormalities to the level of DNA sequence has revealed the genetic causes of many diseases, molecular character- ization of chromosomal aberrations has lagged far behind their discovery . To proceed from cytogenetic observation to gene dis- covery and mechanistic explanation, scientists will need access to a Figure 1 Cytogenetic analyses of sequence-integrated clones. a, Using FISH, fluorescent resource of experimental reagents that effectively integrates the signals are observed at cytogenetic bands (grey) where fragments of a sequence-tagged cytogenetic and sequence maps of the human genome. BAC hybridize (red). b, Clones selected on the basis of band location were used in FISH We describe here the results of a concerted effort to assemble such analyses to map the breakpoint of a translocation involving chromosomes 11 and 19 in a a genome-wide resource of well mapped, large-insert DNA clones. patient with multiple congenital malformations and mental retardation (DGAP012, Each clone has been localized directly to chromosomal band(s) by http://dgap.harvard.edu). Clone CTD-3193o13 spans the breakpoint on chromosome 19; FISH (Fig. 1a) and assigned one or more unique sequence tags, red signal is split between the derivative chomosome 11 and derivative 19 chromosomes which can anchor the clone to the emerging draft sequence. We used and is also present on the normal chromosome 19. The GTG-banded karyotype for this complementary strategies to amass the current set of 8,877 clones. patient is 46,XY,t(11;19)(p11.2;p13.3). Table 1 A cytogenetic resource of FISH-mapped, sequence-tagged clones Number of FISH-mapped clones Connections to the draft squence Type of sequence tag Coverage % discordant Chromosome Accession* STS or gene BAC end Total† Avg. density‡ No. of clones Chrom. Site k % concordant anchored to draft sequence§ ................................................................................................................................................................................................................................................................................................................................................................... 1 868 318 355 1,248 4.9 1,127 2 2 95 2 43 180 189 297 1.2 241 3 2 95 3 128 222 178 308 1.5 233 5 6 90 4 42 253 227 341 1.7 275 7 1 92 5 35 237 168 296 1.6 255 3 4 93 6 653 212 176 909 5.1 801 3 1 96 7 25 254 151 324 1.9 274 2 1 96 8 31 181 161 245 1.6 203 5 3 92 9 208 169 252 384 2.7 324 4 2 94 10 191 302 288 454 3.2 382 4 4 93 11 119 243 225 378 2.7 324 6 2 92 12 109 251 178 304 2.2 266 7 2 91 13 182 101 175 278 2.4 252 3 1 96 14 48 167 167 222 2.1 196 3 2 96 15 109 117 154 224 2.2 189 5 2 94 16 72 237 196 267 2.8 222 4 2 95 17 21 71 77 119 1.3 93 10 1 89 18 9 73 76 105 1.3 86 2 0 98 19 7 55 49 76 1.1 56 14 0 86 20 228 107 112 388 5.6 333 1 1 98 21 4 64 52 85 1.8 72 1 0 99 22 217 123 108 343 6.5 303 3 1 96 X 641 274 150 872 5.5 782 2 2 96 Y 7 13 15 17 0.3 14 7 0 93 Subtotal 3,997 4,224 3,879 8,484 2.6 7,303 3.6 1.9 95 Multiple sites¶ 209 100 220 393 n.a. 297 n.a. n.a. n.a. Total 4,206 4,324 4,099 8,877 n.a. 7,600 ................................................................................................................................................................................................................................................................................................................................................................... All clones are associated with a sequence-tag; localized directly to cytogenetic bands by FISH; BACs, P1, or PACs; archived as single-colony-purified stocks; and publicly available. n.d., not done. n.a., not applicable. * Clones whose draft or finished sequence is deposited in GenBank. † Total is less than sum of preceding three columns because some clones have .1 type of sequence tag. ‡ In clones per Mb, that is, number of FISH-mapped clones/chromosome size in Mb . § Sequence tags of 8,325 single-site and 352 multisite clones were used to search the 7 October 2000 draft. Clones whose sole tag consisted of a Unigene accession (Hs.) and some multisite clones have not yet been evaluated. k Discordant site refers to clones mapped by FISH to location .1 band away from, but on same chromosome as, neighbours on draft. ¶ Because most clones in the resource were not selected randomly, fraction of multi-site clones does not accurately reflect frequency of low-copy duplications in the genome. 954 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature duplicated at more than one location in the genome (to flag regions randomly selected 1,243 clones from this library for FISH analysis. of the genome that might complicate sequence assembly ). The The number of clones assigned to each chromosome correlated well molecular signatures are STSs (many corresponding to genes or with chromosome size, with no significant bias in the distribution of expressed sequence tags (ESTs)), BAC end sequences, or the actual clones between Giemsa (G)-dark and G-light bands of chromo- draft or final sequence of the clone (Table 1). Earlier publications somes (see Supplementary Information 2 and 3). have described genome-wide and chromosome-specific subsets of Cytogenetic mapping is one of several methods that can produce 8–12 this collection . a framework of ordered clones upon which the human sequence can Each clone is publicly available as single-colony-purified bacterial be assembled. The resource provides an opportunity to cross-check stocks and is ready for distribution. Each clone can each be obtained these critical framework maps, because over 3,300 FISH-mapped from one of three stock centres by e-mail: mapped-clones@mail. clones have STSs that reference the radiation hybrid or linkage 15,16 cho.org, [email protected] and [email protected]. The maps . Overall, the concordance between cytogenetic map order website http://www.ncbi.nlm. nih.gov/genome/cyto provides infor- and marker order established by radiation hybrid and linkage mation about all clones in this collection, including how to obtain mapping is very high for clones with single cytogenetic locations each clone. (Additional information can be obtained at the websites (94 – 98%, depending on the map; Table 2). Significant discrepancies listed in Supplementary Information 1). were observed for only around 140 of these clones and are probably The 8,877 clones provide excellent coverage of the human due to errors in clone tracking. Integration of cytogenetic and genome (Table 1), with at least one clone on average per megabase linkage maps also aids efforts to map disease genes. The location of (Mb) for 23 of the 24 chromosomes. Clone density ranges from the cytogenetic abnormality in one patient can guide the choice of greater than ,5 clones per Mb for chromosomes 1, 6, 20, 22 and X polymorphic markers to assess linkage in other families that have to about 0.3 clones per Mb for chromosome Y. similar phenotypes, but no visible chromosomal aberrations. Our study provides an assessment of the representation of the At present, 7,303 clones that map to single cytogenetic locations human genome in the RPCI-11 BAC library , which serves as the are positioned by their sequence tags on the draft sequence assembly intermediate template for most sequencing efforts and the founda- of 7 October 2000 (Table 1). The fraction of clones located on tion of genome-wide contig assembly by fingerprint analyses .We the draft sequence ranges from 76% to 91% across different p13 p12 p11.2 p11.1 q11 q12 q13 q14 q15 q21 q22 q23 q24.1 q24.2 q24.3 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrX chrY 10Mbp 20Mbp 30Mbp 40Mbp 50Mbp 60Mbp 70Mbp 80Mbp 90Mbp 100Mbp 110Mbp 120Mbp 130Mbp 140Mbp Position in draft sequence Figure 2 The correspondence between cytogenetic location and position on the genome.ucsc.edu/goldenPath/mapPlots/. Genome browsers that assist researchers in 7 October 2000 draft sequence for chromosome 12. The band location of each clone is navigating from cytogenetic location to other maps and detailed, annotated sequence indicated by a range on the y-axis. Clones mapping to chromosomes other than 12 are information are available at http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_srch (NCBI indicated at the bottom. Colours differentiate assignments made in different laboratories. Mapviewer, which includes chromosomal aberrations associated with cancer and Each clone is anchored on the draft sequence by one or more sequence tags. Plots for the inherited disorders), http://www.ensembl.org/ and http://genome.ucsc.edu. other chromosomes and the 5 September, 2000 assembly can be found at http:// NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 955 © 2001 Macmillan Magazines Ltd Cytogenic position letters to nature regions encompassing sequence tags of multi-site clones (either the Table 2 Clones connecting the cytogenetic map and other maps of the human genome sequence of the FISH-mapped clone or a surrogate clone from the assembly) contain blocks of homology found at an average of Map type Version Number % concordant % discordant around 3.9 chromosomal locations (compared to around 1.3 for Chrom. Site the regions underlying clones with single FISH signals). The regions Genetic Genethon 1,686 98 1.4 0.7 observed by FISH and revealed through homology searches are not Marshfield 1,757 98 1.4 0.9 fully congruent, however (not shown). These findings indicate that Radiation hybrid GM99-GB4 1,433 98 1.3 1.2 GM99-G3 1,654 96 2.5 1.4 both FISH and sequence analyses may underestimate large-scale TNG 908 94 2.9 2.5 duplications and that these complex, inter-related regions of the Draft sequence 5 Sept. 2000 7,364 94 3.7 2.2 genome will require special attention during the finishing stages of 7 Oct. 2000 7,303 95 3.6 1.9 ............................................................................................................................................................................. genome sequencing. Many clones have markers positioned on more than one map. Only clones assigned to single chromosome locations by FISH are considered above. An additional 91 clones that map by FISH to The extensive integration of cytogenetic and primary sequence more than one location contain STSs placed on other maps. STSs are by definition unique, single- data gives investigators access to fine-structure information — copy markers, so each is assigned to a single genomic location by other mapping approaches. In 88% of these 91 cases, the STS location corresponds to one of the FISH-detected locations. including details on predicted genes— for cytogenetic locations of interest. Tools such as NCBI’s MapViewer and the UCSC and chromosomes (see Supplementary Information 4). We expect these ENSEMBL genome browsers (see Fig. 2 for URLs) allow researchers to navigate readily between chromosomal location and annotated percentages to rise as more sequence is merged into the draft and algorithms for locating tags are refined. sequence. This integration provides insight into the sequence differences The connections between the cytogenetic map and the draft sequence are well distributed across the genome, and the corre- underlying cytogenetic banding patterns. Sequence analyses of 200- spondence in position on the two maps is excellent for these 7,303 kilobase (kb) regions surrounding the sequence tags of 338 clones clones (Fig. 2 shows chromosome 12 as an example). Of the 943 mapped with the finest band resolution reveal more striking contigs of overlapping clones in the 7 October 2000 draft sequence, differences in the base-pair composition between Giemsa-positive 660 are connected to the cytogenetic map by at least one clone, and and -negative bands than were predicted from earlier studies . 531 by two or more clones. Thus, many contigs can be oriented on These clones were mapped with high precision to 850-level bands of varying staining intensity on seven chromosomes. The AT content the chromosome on the basis of FISH results of constituent clones. Relatively few discrepancies between cytogenetic location and of 58 of the 59 clones in the darkest G-bands exceeds the genome- wide average of 0.59 (mean 0.63), whereas the AT content of only 22 position in the draft sequence are apparent at this level of resolution (,5% of the clones map either to other chromosomes or more than of the 143 clones in G-negative bands is higher than average (mean 0.55; x = 43, P , 0.005). These data confirm that dark G-bands are one band away from the expected position; Table 1). We found only eight locations where the cytogenetic data indicated that portions of more AT-rich than G-negative bands. The utility of a sequence-integrated cytogenetic resource is the sequence were misplaced within an earlier draft assembly (5 September 2000). The sequencing centres used these cytogenetic illustrated by two examples. In the first, clones are applied in findings to locate errors in the assembly and produce the later draft conventional FISH assays to rapidly narrow the search for candidate of improved quality (Table 2). genes disrupted or deregulated by translocations causing develop- FISH analyses of this clone collection reveal abundant paralogous mental disorders. The process is expedited by selection of clones relationships among sites dispersed across the human genome. Of assigned to the regions implicated by banding analyses. In a patient 1,243 clones randomly selected from the RPCI-11 library, 5.4% with multiple congenital malformations and mental retardation (DGAP012, http://dgap.harvard.edu), a breakpoint-spanning clone hybridize to more than one chromosomal location (see Supple- mentary Information 3). The entire collection includes 393 clones was identified (Fig. 1b). This clone spans a 170-kb interval contain- ing the gene for MKK7, a human mitogen-activated protein kinase, that together identify over 150 bands containing at least one segment with significant homology to one or more (up to 25) and a novel sequence with homology to the tre-2 oncogene, both plausible candidate genes. More typically, breakpoints will be other sites in the genome (see Supplementary Information 5). These data provide clues to duplications and exchanges that have occurred mapped to an interval between neighbouring clones. For example, within and between chromosomes. Among the 393 clones, 111 a translocation implicated in mental retardation in another patient contain blocks duplicated within the same chromosome; 282 maps to an interval containing at least 12 genes, including proto- hybridize to more than one chromosome. Paralogous relationships cadherin 8, a promising candidate given its exclusive expression in involving pericentromeric and subtelomeric regions of multiple fetal and adult brain . chromosomes are particularly frequent and complex. Clones in the In the second example, an array of around 2,000 BAC clones from collection also identify low-copy duplications specific to chromo- the collection is used to perform a genome-wide scan for segmental somes 1, 7, 11 and 16, the pseudoautosomal regions of X and Y, and aneuploidy by comparative genomic hybridization (CGH) (Fig. 3 and A. Snijders et al., in preparation). The array format offers better sites of the olfactory receptor gene family . Many previously 21,22 undescribed patterns were also observed; some were confirmed sensitivity and resolution than metaphase chromosomes, the traditional target for CGH , and, because the arrayed clones are with two or more clones, but others require further study to verify that they reflect true duplications. integrated into the draft, copy-number abnormalities can be related directly to sequence information. To illustrate the power of array Many of these duplications are functionally significant, as some have generated multigene families, and some are potential sites of CGH, the ML-2 cancer cell line was ‘karyotyped’ using the array. recombination events, which can result in chromosome abnormal- Array CGH revealed relative copy-number losses on 1p, 6q, 11q and ities. The cytogenetic data should greatly facilitate analyses of these 20p and gains of 12, 13 and 20q (Fig. 3). Copy-number abnorm- regions, which are likely to pose challenges to sequence assembly. alities on chromosomes 6, 11 and 20 were subsequently confirmed The sequence tags of 84% of the clones that hybridize to more than by FISH using clones predicted by array CGH to be included in the one site were placed in the 7 October 2000 draft assembly, and the region of loss. Several of these alterations were noted in previous location(s) were roughly consistent with at least one FISH observa- banding analyses (1p−,6q−, 11q−, +12, +13q+) , but array CGH tion for 88% of these clones. Collectively, the multisite clones locates the breakpoints precisely relative to BACs that reference specific locations in the sequence. highlight regions that are more likely to become entangled with other regions of the genome during sequence assembly than clones More than 7,500 clones now link the cytogenetic map and sequence of the human genome. Application of these reagents in with single FISH locations. Indeed, global BLAST analyses show that 956 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature 20q 1p 6q 11q 20p 1p Xq Position in genome 2.0 2.0 b c 1.5 1.5 1.0 1.0 0.5 0.5 0 0 0 1,000 2,000 3,000 4,000 5,000 6,000 0 10 20304050 60 70 80 90 Order on G3-radiation hybrid map Position on Genethon linkage map (cM) Figure 3 Copy-number analysis of myeloblastic leukaemia ML-2 cell line using CGH and a BAC normalized to the median ratio for all 2,000 clones on the array, ordered from 1pter genome-wide array of around 2,000 BAC clones. The ML-2 cell line has acquired to Xqter. Arrows, chromosomal regions showing significant copy number variations. The chromosomal abnormalities in addition to those present in the original tumour during lower ratio on the X indicates expected ratio for mismatched sex of test and reference long-term culture. CGH maps regions of abnormal copy number by comparing the relative DNAs. Fluorescence ratios of clones on chromosomes 11 (b) and 20 (c) are shown with efficiency with which test (Cy3-labelled ML-2 DNA) and reference (Cy5-labelled normal clones ordered according to position of their STSs on the G3 radiation hybrid or Genethon female DNA) hybridize to clones on the array. The array excludes clones that hybridize to linkage maps, respectively. multiple sites in the genome. a, Fluorescence ratios of Cy3 to Cy5 fluorescence for each 8. Cheung, V. G. et al. A resource of mapped human bacterial artificial chromosome clones. Genome Res. combination with increasingly detailed knowledge of genes and 9, 989 – 993 (1999). other functional motifs in the human sequence will transform the 9. Korenberg, J. R. et al. Human genome anatomy: BACs integrating the genetic and cytogenetic maps process of identifying genes that are altered in cancer and other for bridging genome and biomedicine. Genome Res 9, 994 – 1001 (1999). 10. Leversha, M. A., Dunham, I. & Carter, N. P. A molecular cytogenetic clone resource for chromosome diseases. Ultimately, this resource will contribute to a better under- 22. Chromosome Res. 7, 571– 573 (1999). standing of the organization of the cell nucleus, the compacting of 11. Kirsch, I. R. et al. A systematic, high-resolution linkage of the cytogenetic and physical maps of the DNA into mitotic chromosomes, and the basis of the chromosomal human genome. Nature Genet. 24, 339 – 340 (2000). banding patterns that have been so valuable in uncovering the 12. Kirsch, I. R. & Ried, T. Integration of cytogenetic data with genome maps and available probes: Present status and future promise. Semin. Hematol. 37, 420 – 428 (2000). aetiology of human diseases. M 13. Osoegawa, K. et al. Bacterial artificial chromosome library for sequencing the human genome. Genome Res. (in the press). Methods 14. Olivier, M. et al. A high resolution radiation hybrid map of the human genome draft sequence. Science GenBank was screened for draft, finished or end sequences derived from clones in this (in the press). collection. BACs were screened for STS content by a combination of hybridization and 15. Yu, A. et al. Comparison of human genetic and sequence-hosed physical maps. Nature 409, 951 – 953 polymerase chain reaction (see refs 8, 25 and Supplementary Information for details). (2001). 16. Dib, C. et al. A comprehensive genetic map of the human genome based on 5,264 microsatellites. Sequence tags were located on the draft sequence by a combination of methods (see Supplementary Information and refs 26, 27). Sequence at these locations was compiled Nature 380, 152 – 154 (1996). 17. Trask, B. J. et al. Large multi-chromosomal duplications encompass many members of the olfactory with the results of a genome-wide BLAST analysis (ref. 2 and J. A. Bailey and E. E. Eichler, receptor gene family in the human genome. Hum. Mol. Genet. 7, 2007– 2020 (1998). in preparation) to identify paralogous regions of the genome (regions in the draft 18. Saccone, S. et al. Correlations between isochores and chromosomal bands in the human genome. Proc. sequence containing , 20 kb of sequence that match sequence of the FISH-mapped clone Natl. Acad Sci. USA 90, 11929– 11933 (1993). or that of a surrogate clone from the assembly at , 90% identity in non-repeat-masked 19. Mitelman, F. (Ed.) ISCN (1995): An International System for Human Cytogenetics Nomenclature bases over each 1-kb segment), and these locations were translated into estimated band (S. Karger, Basel, 1995). positions using a dynamic programming algorithm (T. S. Furey et al., in preparation; and 20. Strehl, S. et al. Characterization of two novel protocadherins (PCDH8 and PCDH9) localized on see Supplementary Information). 4,28 human chromosome 13 and mouse chromosome 14. Genomics 53, 81 – 89 (1998). Details of FISH procedures are provided elsewhere . Only locations of unique or low- 21. Pinkel, D. et al. High resolution analysis of DNA copy number variation using comparative genomic copy portions of the clone are identified, because high-copy interspersed repetitive hybridization to microarrays. Nature Genet. 20, 207 – 211 (1998). sequences were suppressed by addition of unlabelled Cot1 DNA. Replicate analyses 22. Solinas-Toldo, S. et al. Matrix-based comparative genomic hybridization: biochips to screen for indicate that the precision of FISH assignments to metaphase bands is roughly 5 – 10 Mb genomic imbalances. Genes Chromosom. Cancer 20, 399 – 407 (1997). (1 – 1.5 band). A subset of 442 clones was ordered at very high (,2 – 3-Mb) resolution . 23. Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid FISH analyses were performed using DNA from the bacterial stock used for STS typing. tumors. Science 258, 818 – 821 (1992). Data that failed to replicate (for example, replicate FISH analyses of the same clone or 24. Ohyashiki, K., Ohyashiki, J. H. & Sandberg, A. V. Cytogenetic characterization of putative human different clones assigned the same marker) have been removed. Hybridization to arrays myeloblastic leukemia cell lines (ML-1, -2, and -3): origin of the cells. Cancer Res. 46, 3642–3647 (1986). was carried out as described previously and by Snijders et al. (in preparation). 25. Morley, M. GenMapDB: A database of mapped human BAC clones. Nucleic Acids Res. 29, 144 – 147 (2001). Received 7 November 1999; accepted 20 December 2000. 26. Schuler, G. D. Electronic PCR: bridging the gap between genome mapping and genome sequencing. 1. The International Human Genome Mapping Consortium. A physical map of the human genome. Trends Biotechnol. 16, 456 – 459 (1998). Nature 409, 934 – 941 (2001). 27. Zhang, Z., Schwartz, S., Wagner, L. & Miller, W. A greedy algorithm for aligning DNA sequences. 2. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human J. Comput. Biol. 7, 203 – 214 (2000). genome. Nature 409, 860 – 921 (2001). 28. Korenberg, J. R., Chen, X. -N. Human cDNA mapping using a high-resolution R-banding technique 3. Caspersson, T. et al. Chemical differentiation along metaphase chromosomes. Exp. Cell Res. 49, 219 – and fluorescence in situ hybridization. Cytogenet. Cell Genet. 69, 196 – 200 (1995). 222 (1968). 29. Albertson, D. G. et al. Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a 4. Trask, B. J. in Genome Analysis: A Laboratory Manual Vol. 4, 303 – 413 (Cold Spring Harbor Laboratory candidate oncogene. Nature Genet. 25, 144 – 146 (2000). Press, Cold Spring Harbor, New York, 1999). 30. Trask, B. J., van den Engh, G., Mayall, B. & Gray, J. W. Chromosome heteromorphism quantified by 5. Collins, F. S. Positional cloning moves from perditional to traditional. Nature Genet. 9, 347– 350 high resolution bivariate flow karyotyping. Am. J. Hum. Genet. 45, 738 – 752 (1989). (1995). 6. Mitelman, F. Catalog of Chromosome Aberrations in Cancer (Wiley, New York, 1998). 7. Eichler, E. E. Masquerading repeats: paralogous pitfalls of the human genome. Genome Res. 8, 758 – Supplementary information is available from Nature’s World-Wide Web site 762 (1998). (http://www.nature/com) or as paper copy from the London editorial office of Nature. NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 957 © 2001 Macmillan Magazines Ltd Ratio Ratio letters to nature Acknowledgements Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA; 6, Departments of Pediatrics and Human Genetics, Cedars-Sinai Medical Center, We thank M. Arcaro, M. Bakis, J. Burdick, J. Chang, H.-C. Chen, S. Chiu, Y. Fan, C. Harris, L. Haley, R. Hosseini, J. Kent, M. A. Leversha, J. Martin, L.-T. Nguyen, P. Quinn, Y. H. 8700 Beverly Boulevard, Los Angeles, California 90048, USA; 7, Computer Science Ramsey, T. Reppert, L. J. Rogers, J. Shreve, J. Stalica, M. Wang, T. Weber, A. M. Yavor, J. Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, Young, K. Zatloukal, and members of the TIGR BAC Ends Team for assistance. This work California 95064-1077, USA; 8, Department of Biology, California Institute of was supported by grants from NIH (NCI, NHGRI, NIDCD and NICHD), US DOE, NSF, Technology, Mail Code 147-75, Pasadena, California 91125, USA; 9, University of HHMI, PPG, Merck Genome Research Institute, Vysis, Inc., and start-up funds provided California San Francisco Cancer Center, Box 0808, San Francisco, California by Obstetrics and Gynecology at Brigham and Women’s Hospital. 94143-0808, USA; 10, Stanford University, Genome Lab, Mail Code 5120, Correspondence should be addressed to B.J.T. Stanford, California 94305-5120, USA; 11, Sanger Center, Wellcome Trust (e-mail: [email protected]). Genome Campus, Hinxton, Cambridge, CB10 1SA, UK; 12, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North C3-168, P.O. Box 19024, The BAC Resource Consortium Seattle, Washington 98109-1024, USA; 13, Department of Molecular 1 2 3 4 5 6 Biotechnology, University of Washington, Box 357730, Seattle, Washington 98195- V. G. Cheung *, N. Nowak *, W. Jang , I. R. Kirsch , S. Zhao , X.-N. Chen , 7 8 9 10 2 7730, USA; 14, Departments of Obstetrics and Gynecology and Pathology, T. S. Furey , U.-J. Kim †, W.-L. Kuo , M. Olivier , J. Conroy , 11 12 4 2 13 Brigham and Women’s Hospital, Amory Lab Building 3rd floor, Boston, A. Kasprzyk , H. Massa , R. Yonescu , S. Sait , C. Thoreen †, 9 14 15 1 11 Massachusetts 02115, USA; 15, Department of Human Genetics, Case Western A. Snijders , E. Lemyre , J. A. Bailey , A. Bruzel , W. D. Burrill , 11 13 11 12 16 Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA; S. M. Clegg , S. Collins , P. Dhami , C. Friedman ,C.S.Han , 14 8 14 17 1 16, Joint Genome Institute-Los Alamos National Laboratory, MS M888 B-N1, S. Herrick , J. Lee , A. H. Ligon , S. Lowry , M. Morley , 1 2,18 17 17 P.O. Box 1663, Los Alamos, New Mexico 87545, USA; 17, Joint Genome Institute- S. Narasimhan , K. Osoegawa , Z. Peng , I. Plajzer-Frick , 14 17 3 11 9 Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 84-171, B. J. Quade , D. Scott , K. Sirotkin , A. A. Thorpe , J. W. Gray , 19 9 4 20 4 Berkeley, California 94720, USA; 18, Children’s Hospital Oakland Research J. Hudson , D. Pinkel , T. Ried , L. Rowen , G. L. Shen-Ong , 4 11 21 17 10 Institute, 747 52nd Street, Oakland, California 94609, USA; 19, Research R. L. Strausberg , E. Birney , D. F. Callen , J.-F. Cheng , D. R. Cox , 16 11 15 22 Genetics, 2130 Memorial Parkway, Huntsville, Alabama 35801, USA; N. A. Doggett , N. P. Carter , E. E. Eichler , D. Haussler , 6 14 9 3 20, Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, J. R. Korenberg , C. C. Morton , D. Albertson , G. Schuler ,P. J. de 2,18 12 Washington 98105-6099, USA; 21, Department of Cytogenetics and Molecular Jong & B. J. Trask Genetics, Women’s and Children’s Hospital, 72 King William Road, * These authors contributed equally to this work. North Adelaide, South Australia 5006, Australia; 22, Howard Hughes Medical 1, Department of Pediatrics, University of Pennsylvania, The Children’s Hospital Institute, Computer Science Department, University of California Santa Cruz, of Philadelphia, 3516 Civic Center Boulevard, ARC 516, Philadelphia, 1156 High Street, Santa Cruz, California 95064– 1077, USA Pennsylvania 19104, USA; 2, Roswell Park Cancer Institute, Elm and Carleton Present addresses: PanGenomics, 6401 Foothill Boulevard, Tujunga, Street, Buffalo, New York 14263, USA; 3, National Center for Biotechnology California 91024, USA (U.-J.K.); Harvard Medical School, 240 Longwood Information, National Library of Medicine, Building 38A/Room 8N805, Avenue, Cell Biology, Cambridge, Massachusetts 02115, USA (C.T.); Gene Logic, Bethesda, Maryland 20894, USA; 4, National Cancer Institute, NIH, Building 10/ Inc., 708 Quince Orchard Road, Gaithersburg, Maryland 20878, USA Room 12N214, Bethesda, Maryland 20889-5105, USA; 5, The Institute for (G.L.S.-O.). 958 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Springer Journals

Integration of cytogenetic landmarks into the draft sequence of the human genome

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Springer Journals
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Copyright © 2001 by Macmillan Magazines Ltd.
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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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0028-0836
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1476-4687
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10.1038/35057192
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letters to nature 11. Mohrenweiser, H. W., Tsujimoto, S., Gordon, L. & Olsen, A. S. Regions of sex-specific hypo- and low recombination. Nucleotide and haplotype diversity will also hyper-recombination identified through integration of 180 genetic markers into the metric physical probably parallel recombination rates. Although our baseline long- map of human chromosome 19. Genomics 47, 153 – 162 (1998). range recombination rates will be useful, they should be recalculated 12. Nicolas, A. Relationship between transcription and initiation of meiotic recombination: toward when the human genomic sequences are finished and as higher chromatin accessibility. Proc. Natl Acad. Sci. USA 95, 87– 89 (1998). 13. Wahls, W. P. Meiotic recombination hotspots: shaping the genome and insights into hypervariable resolution genetic maps become available. In the more distant minisatellite DNA change. Curr. Top. Dev. Biol. 37, 37 – 75, (1998). future, genotyping greater numbers of reference families at much 14. Faris, J. D., Haen, K. M. & Gill, B. S. Saturation mapping of a gene-rich recombination hot spot region higher polymorphism densities will lead to short-range maps of in wheat. Genetics 154, 823 – 835 (2000). 15. Kliman, R. M. & Hey, J. Reduced natural selection associated with low recombination in Drosophila recombination hot spots. M melanogaster. Mol. Biol. Evol. 10, 1239– 1258 (1993). 16. Chen, T. L. & Manuelidis, L. SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size. Methods Chromosoma 98, 309 – 316 (1989). 17. Payseur, B. A. & Nachman, M. W. Microsatellite variation and recombination rate in the human Connection of genetic and physical maps genome. Genetics 156, 1285– 1298 (2000). We used short, single-pass genomic sequences and/or PCR primer sequences for STRPs to 18. Nachman, M. W., Bauer, V. L., Crowell, S. L. & Aquadro, C. F. DNA variability and recombination rates identify draft or finished bacterial artificial chromosome (BAC) or cosmid sequences at X-linked loci in humans. Genetics 150, 1133– 1141 (1998). 27 28 within GenBank that encompass the STRPs using BLAST and ePCR . Blast criteria were 19. Nachman, M. W. & Crowell, S. L. Contrasting evolutionary histories of two introns of the Duchenne −50 score (bits) . 200, expect (E) value , e , and ratio of matched bases to marker sequence muscular dystrophy gene, Dmd, in humans. Genetics 155, 1855 – 1864 (2000). length . 85%. ePCR criteria were no more than one base mismatch in each primer and 20. Majewski, J. & Ott, J. GT repeats are associated with recombination on human chromosome 22. size of PCR product within allele size range for the STRP. About 75% of the STRPs were Genome Res. 10, 1108– 1114 (2000). connected to the long genomic sequences. The reasons for failure of the remaining 25% are 21. Bernardi, G. Isochores and the evolutionary genomics of vertebrates. Gene 241, 3 – 17 (2000). not fully understood, but include absence of the corresponding sequence in GenBank and 22. Eisenbarth, I., Vogel, G., Krone, W., Vogel, W. & Assum, G. An isochore transition in the NF1 gene poor quality of the STRP sequences. As the genetic maps are marker rich, the absence of region coincides with a switch in the extent of linkage disequilibrium. Am. J. Hum. Genet. 67, 873 – 880 25% was not a serious limitation. Tables of STRPs with GenBank sequence accession (2000). numbers for encompassing BACs, genetic map positions and recombination rates are 23. Yu, J. et al. Individual variation in recombination among human males. Am. J. Hum. Genet. 59, 1186– available from the Marshfield web site. 1192 (1996). 24. Lien, S., Szyda, J., Schechinger, B., Rappold, G. & Arnheim, N. Evidence for heterogeneity in recombination in the human pseudoautosomal region: High resolution analysis by sperm typing and Determination of recombination rates radiation-hybrid mapping. Am. J. Hum. Genet. 66, 557 – 566 (2000). For each sequence assembly we built new female, male and sex-average genetic maps, using 25. Carrington, M. Recombination within the human MHC. Immunol Rev. 167, 245– 256 (1999). the marker order provided by the assemblies and using the genotyping data from the eight 26. Jeffreys, A. J., Ritchie, A. & Neumann, R. High resolution analysis of haplotype diversity and meiotic CEPH reference families . We fitted cubic splines to plots of genetic versus physical crossover in the human TAP2 recombination hot spot. Hum. Mol. Genet. 9, 725 – 733 (2000). distance, and from these curves we obtained recombination rates as first derivatives . The 27. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search statistical significance of the recombination rates was estimated by computer simulation of programs. Nucleic Acids Res. 25, 3389 (1997). 1,000 iterations of recombination within each interval between markers, assuming a 28. Schuler, G. D. Sequence mapping by electronic PCR. Genome Res. 7, 541 – 550 (1997). constant level of recombination across the genome for each sex. The constant levels of 29. Zhao, C., Heil, J., & Weber, J. L. A genome wide portrait of short tandem repeats. Am. J. Hum. Genet. recombination were taken as the total genetic lengths of all the assemblies analysed divided 65, (Suppl.) A102 (1999). by the total physical lengths of these assemblies. 30. Huttley, G., A., Smith, M. W., Carrington, M. & O’Brien, S. J. A scan for linkage disequilibrium across the human genome. Genetics 152, 1711– 1722 (1999). Computation of marker and sequence parameters Supplementary information is available from Nature’s World-Wide Web site We calculated STRP heterozygosities using genotypes of individuals within the eight (http://www.nature.com) or as paper copy from the London editorial office of Nature, and CEPH families. We obtained STRP positions relative to centromeres and telomeres as the from the Marshfield Web site (http://research.marshfieldclinic.org/genetics). fractional sex-average genetic map distances from the centromeres to the telomeres (value of 0 for a STRP at the centromere and 1.0 for a STRP at the telomere) . GC content and Acknowledgements STR densities were obtained from programs written and tested at Marshfield . STR densities were measured as numbers of runs of non-interrupted repeats rather than total This work was supported by contracts from the US National Institutes of Health and numbers of repeats. Minimum values of n for (A) ,(AC) ,(AGAT) , (AAN) and n n n n Department of Energy. Assistance was also provided by the chromosome 6 and 20 project (AAAN) sequences were 12, 11 or 19 ((AC) ), 5, 7 and 5, respectively. We obtained n n groups at the Sanger Centre, supported by the Wellcome Trust. interspersed repetitive element densities using the program Repeat Masker Correspondence and requests for materials should be addressed to J.L.W. (http://ftp.genome.washington.edu/RM/RepeatMasker.html). SINEs and LINEs were (e-mail: [email protected]fldclin.edu). defined by Repeat Masker and consist primarily of Alu and L1 elements, respectively. We computed all DNA sequence parameters over 250-kb windows centred about each STRP. For markers # 125 kb from the ends of the sequence assemblies, we defined the window as the 125 kb of proximal sequence plus all available distal sequence. Unknown bases in the sequence assemblies were excluded from analysis. All parameters were corrected for reduced window size owing to unknown bases or proximity to ends. ................................................................. Measurement of linkage disequilibrium Integration of cytogenetic landmarks Recombination deserts and jungles were selected as those chromosomal regions with sex- average recombination rates of ,0.3 or .3.0, respectively. We measured linkage disequili- into the draft sequence of the human brium for all pairs of STRPs within the deserts (449 pairs) and jungles (467 pairs) using Fisher’s exact test . Only disequilibrium results that were significant at P # 0.01 were plotted genome in Fig. 2. An overall P-value was obtained by a permutation test treating the regions as units in order to account for the dependence between marker pairs within a region. The BAC Resource Consortium* Received 27 October; accepted 8 December 2000. 1. The BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the * Authorship of this paper should be cited using the names of authors that appear at human genome. Nature 409, 953 – 958 (2001). the end. 2. The International Human Genome Mapping Consortium. A physical map of the human genome. Nature 409, 934 – 941 (2001). ................. ......................... ......................... ......................... ......................... ......................... 3. Deloukas, P. et al. A physical map of 30,000 human genes. Science 282, 744 – 746 (1998). We have placed 7,600 cytogenetically defined landmarks on the 4. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human draft sequence of the human genome to help with the character- genome. Nature 409, 860 – 921 (2001). 5. Broman, K. W., Murray, J. C., Sheffield, V. C., White, R. L. & Weber, J. L. Comprehensive human genetic ization of genes altered by gross chromosomal aberrations that maps: Individual and sex-specific variation in recombination. Am. J. Hum. Genet. 63, 861–869 (1998). cause human disease. The landmarks are large-insert clones 6. Dunham, I. et al. The DNA sequence of human chromosome 22. Nature 402, 489 – 495 (1999). mapped to chromosome bands by fluorescence in situ hybridiza- 7. Hattori, M. et al. The DNA sequence of human chromosome 21. Nature 405, 311 – 319 (2000). tion. Each clone contains a sequence tag that is positioned on the 8. Fain, P. R., Kort, E. N., Yousry, C., James, M. R. & Litt, M. A high resolution CEPH crossover mapping panel and integrated map of chromosome 11. Hum. Mol. Genet. 5, 1631 – 1636 (1996). genomic sequence. This genome-wide set of sequence-anchored 9. Bouffard, G. G. et al. A physical map of human chromosome 7: An integrated YAC contig map with clones allows structural and functional analyses of the genome. average STS spacing of 79 kb. Genome Res. 7, 673 – 692 (1997). This resource represents the first comprehensive integration of 10. Nagaraja, R. et al. X chromosome map at 75-kb STS resolution, revealing extremes of recombination cytogenetic, radiation hybrid, linkage and sequence maps of the and GC content. Genome Res. 7, 210 – 222 (1997). NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 953 © 2001 Macmillan Magazines Ltd letters to nature human genome; provides an independent validation of the The set, which consists primarily of bacterial artificial chromosome 1,2 sequence map and framework for contig order and orientation; (BAC) clones, includes clones targeted to contain sequence-tagged surveys the genome for large-scale duplications, which are likely sites (STSs) ordered along the genome by genetic linkage or radiation to require special attention during sequence assembly; and allows hybrid mapping (for well ordered and distributed coverage); clones a stringent assessment of sequence differences between the dark randomly selected for end sequencing from the RPCI-11 library (for and light bands of chromosomes. It also provides insight into coverage of regions low in STSs); clones identified during intense large-scale chromatin structure and the evolution of chromo- mapping efforts that preceded sequencing of some chromosomes somes and gene families and will accelerate our understanding of (for denser coverage); and clones suspected of being partially the molecular bases of human disease and cancer. With the draft of the human genome available , scientists can conduct global analyses of its gene content, structure, function and ab variation. One important challenge is to define the genetic con- tribution to human diseases. For many developmental disorders, inherited conditions and cancers, gross chromosomal aberrations provide clues to the locations of the causative molecular defects. nl19 These aberrations are visible as alterations in chromosomal banding der(11) patterns or in the number or relative positions of DNA sequences der(19) labelled by fluorescence in situ hybridization (FISH) . Although tracing gross abnormalities to the level of DNA sequence has revealed the genetic causes of many diseases, molecular character- ization of chromosomal aberrations has lagged far behind their discovery . To proceed from cytogenetic observation to gene dis- covery and mechanistic explanation, scientists will need access to a Figure 1 Cytogenetic analyses of sequence-integrated clones. a, Using FISH, fluorescent resource of experimental reagents that effectively integrates the signals are observed at cytogenetic bands (grey) where fragments of a sequence-tagged cytogenetic and sequence maps of the human genome. BAC hybridize (red). b, Clones selected on the basis of band location were used in FISH We describe here the results of a concerted effort to assemble such analyses to map the breakpoint of a translocation involving chromosomes 11 and 19 in a a genome-wide resource of well mapped, large-insert DNA clones. patient with multiple congenital malformations and mental retardation (DGAP012, Each clone has been localized directly to chromosomal band(s) by http://dgap.harvard.edu). Clone CTD-3193o13 spans the breakpoint on chromosome 19; FISH (Fig. 1a) and assigned one or more unique sequence tags, red signal is split between the derivative chomosome 11 and derivative 19 chromosomes which can anchor the clone to the emerging draft sequence. We used and is also present on the normal chromosome 19. The GTG-banded karyotype for this complementary strategies to amass the current set of 8,877 clones. patient is 46,XY,t(11;19)(p11.2;p13.3). Table 1 A cytogenetic resource of FISH-mapped, sequence-tagged clones Number of FISH-mapped clones Connections to the draft squence Type of sequence tag Coverage % discordant Chromosome Accession* STS or gene BAC end Total† Avg. density‡ No. of clones Chrom. Site k % concordant anchored to draft sequence§ ................................................................................................................................................................................................................................................................................................................................................................... 1 868 318 355 1,248 4.9 1,127 2 2 95 2 43 180 189 297 1.2 241 3 2 95 3 128 222 178 308 1.5 233 5 6 90 4 42 253 227 341 1.7 275 7 1 92 5 35 237 168 296 1.6 255 3 4 93 6 653 212 176 909 5.1 801 3 1 96 7 25 254 151 324 1.9 274 2 1 96 8 31 181 161 245 1.6 203 5 3 92 9 208 169 252 384 2.7 324 4 2 94 10 191 302 288 454 3.2 382 4 4 93 11 119 243 225 378 2.7 324 6 2 92 12 109 251 178 304 2.2 266 7 2 91 13 182 101 175 278 2.4 252 3 1 96 14 48 167 167 222 2.1 196 3 2 96 15 109 117 154 224 2.2 189 5 2 94 16 72 237 196 267 2.8 222 4 2 95 17 21 71 77 119 1.3 93 10 1 89 18 9 73 76 105 1.3 86 2 0 98 19 7 55 49 76 1.1 56 14 0 86 20 228 107 112 388 5.6 333 1 1 98 21 4 64 52 85 1.8 72 1 0 99 22 217 123 108 343 6.5 303 3 1 96 X 641 274 150 872 5.5 782 2 2 96 Y 7 13 15 17 0.3 14 7 0 93 Subtotal 3,997 4,224 3,879 8,484 2.6 7,303 3.6 1.9 95 Multiple sites¶ 209 100 220 393 n.a. 297 n.a. n.a. n.a. Total 4,206 4,324 4,099 8,877 n.a. 7,600 ................................................................................................................................................................................................................................................................................................................................................................... All clones are associated with a sequence-tag; localized directly to cytogenetic bands by FISH; BACs, P1, or PACs; archived as single-colony-purified stocks; and publicly available. n.d., not done. n.a., not applicable. * Clones whose draft or finished sequence is deposited in GenBank. † Total is less than sum of preceding three columns because some clones have .1 type of sequence tag. ‡ In clones per Mb, that is, number of FISH-mapped clones/chromosome size in Mb . § Sequence tags of 8,325 single-site and 352 multisite clones were used to search the 7 October 2000 draft. Clones whose sole tag consisted of a Unigene accession (Hs.) and some multisite clones have not yet been evaluated. k Discordant site refers to clones mapped by FISH to location .1 band away from, but on same chromosome as, neighbours on draft. ¶ Because most clones in the resource were not selected randomly, fraction of multi-site clones does not accurately reflect frequency of low-copy duplications in the genome. 954 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature duplicated at more than one location in the genome (to flag regions randomly selected 1,243 clones from this library for FISH analysis. of the genome that might complicate sequence assembly ). The The number of clones assigned to each chromosome correlated well molecular signatures are STSs (many corresponding to genes or with chromosome size, with no significant bias in the distribution of expressed sequence tags (ESTs)), BAC end sequences, or the actual clones between Giemsa (G)-dark and G-light bands of chromo- draft or final sequence of the clone (Table 1). Earlier publications somes (see Supplementary Information 2 and 3). have described genome-wide and chromosome-specific subsets of Cytogenetic mapping is one of several methods that can produce 8–12 this collection . a framework of ordered clones upon which the human sequence can Each clone is publicly available as single-colony-purified bacterial be assembled. The resource provides an opportunity to cross-check stocks and is ready for distribution. Each clone can each be obtained these critical framework maps, because over 3,300 FISH-mapped from one of three stock centres by e-mail: mapped-clones@mail. clones have STSs that reference the radiation hybrid or linkage 15,16 cho.org, [email protected] and [email protected]. The maps . Overall, the concordance between cytogenetic map order website http://www.ncbi.nlm. nih.gov/genome/cyto provides infor- and marker order established by radiation hybrid and linkage mation about all clones in this collection, including how to obtain mapping is very high for clones with single cytogenetic locations each clone. (Additional information can be obtained at the websites (94 – 98%, depending on the map; Table 2). Significant discrepancies listed in Supplementary Information 1). were observed for only around 140 of these clones and are probably The 8,877 clones provide excellent coverage of the human due to errors in clone tracking. Integration of cytogenetic and genome (Table 1), with at least one clone on average per megabase linkage maps also aids efforts to map disease genes. The location of (Mb) for 23 of the 24 chromosomes. Clone density ranges from the cytogenetic abnormality in one patient can guide the choice of greater than ,5 clones per Mb for chromosomes 1, 6, 20, 22 and X polymorphic markers to assess linkage in other families that have to about 0.3 clones per Mb for chromosome Y. similar phenotypes, but no visible chromosomal aberrations. Our study provides an assessment of the representation of the At present, 7,303 clones that map to single cytogenetic locations human genome in the RPCI-11 BAC library , which serves as the are positioned by their sequence tags on the draft sequence assembly intermediate template for most sequencing efforts and the founda- of 7 October 2000 (Table 1). The fraction of clones located on tion of genome-wide contig assembly by fingerprint analyses .We the draft sequence ranges from 76% to 91% across different p13 p12 p11.2 p11.1 q11 q12 q13 q14 q15 q21 q22 q23 q24.1 q24.2 q24.3 chr1 chr2 chr3 chr4 chr5 chr6 chr7 chr8 chr9 chr10 chr11 chr12 chr13 chr14 chr15 chr16 chr17 chr18 chr19 chr20 chr21 chr22 chrX chrY 10Mbp 20Mbp 30Mbp 40Mbp 50Mbp 60Mbp 70Mbp 80Mbp 90Mbp 100Mbp 110Mbp 120Mbp 130Mbp 140Mbp Position in draft sequence Figure 2 The correspondence between cytogenetic location and position on the genome.ucsc.edu/goldenPath/mapPlots/. Genome browsers that assist researchers in 7 October 2000 draft sequence for chromosome 12. The band location of each clone is navigating from cytogenetic location to other maps and detailed, annotated sequence indicated by a range on the y-axis. Clones mapping to chromosomes other than 12 are information are available at http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_srch (NCBI indicated at the bottom. Colours differentiate assignments made in different laboratories. Mapviewer, which includes chromosomal aberrations associated with cancer and Each clone is anchored on the draft sequence by one or more sequence tags. Plots for the inherited disorders), http://www.ensembl.org/ and http://genome.ucsc.edu. other chromosomes and the 5 September, 2000 assembly can be found at http:// NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 955 © 2001 Macmillan Magazines Ltd Cytogenic position letters to nature regions encompassing sequence tags of multi-site clones (either the Table 2 Clones connecting the cytogenetic map and other maps of the human genome sequence of the FISH-mapped clone or a surrogate clone from the assembly) contain blocks of homology found at an average of Map type Version Number % concordant % discordant around 3.9 chromosomal locations (compared to around 1.3 for Chrom. Site the regions underlying clones with single FISH signals). The regions Genetic Genethon 1,686 98 1.4 0.7 observed by FISH and revealed through homology searches are not Marshfield 1,757 98 1.4 0.9 fully congruent, however (not shown). These findings indicate that Radiation hybrid GM99-GB4 1,433 98 1.3 1.2 GM99-G3 1,654 96 2.5 1.4 both FISH and sequence analyses may underestimate large-scale TNG 908 94 2.9 2.5 duplications and that these complex, inter-related regions of the Draft sequence 5 Sept. 2000 7,364 94 3.7 2.2 genome will require special attention during the finishing stages of 7 Oct. 2000 7,303 95 3.6 1.9 ............................................................................................................................................................................. genome sequencing. Many clones have markers positioned on more than one map. Only clones assigned to single chromosome locations by FISH are considered above. An additional 91 clones that map by FISH to The extensive integration of cytogenetic and primary sequence more than one location contain STSs placed on other maps. STSs are by definition unique, single- data gives investigators access to fine-structure information — copy markers, so each is assigned to a single genomic location by other mapping approaches. In 88% of these 91 cases, the STS location corresponds to one of the FISH-detected locations. including details on predicted genes— for cytogenetic locations of interest. Tools such as NCBI’s MapViewer and the UCSC and chromosomes (see Supplementary Information 4). We expect these ENSEMBL genome browsers (see Fig. 2 for URLs) allow researchers to navigate readily between chromosomal location and annotated percentages to rise as more sequence is merged into the draft and algorithms for locating tags are refined. sequence. This integration provides insight into the sequence differences The connections between the cytogenetic map and the draft sequence are well distributed across the genome, and the corre- underlying cytogenetic banding patterns. Sequence analyses of 200- spondence in position on the two maps is excellent for these 7,303 kilobase (kb) regions surrounding the sequence tags of 338 clones clones (Fig. 2 shows chromosome 12 as an example). Of the 943 mapped with the finest band resolution reveal more striking contigs of overlapping clones in the 7 October 2000 draft sequence, differences in the base-pair composition between Giemsa-positive 660 are connected to the cytogenetic map by at least one clone, and and -negative bands than were predicted from earlier studies . 531 by two or more clones. Thus, many contigs can be oriented on These clones were mapped with high precision to 850-level bands of varying staining intensity on seven chromosomes. The AT content the chromosome on the basis of FISH results of constituent clones. Relatively few discrepancies between cytogenetic location and of 58 of the 59 clones in the darkest G-bands exceeds the genome- wide average of 0.59 (mean 0.63), whereas the AT content of only 22 position in the draft sequence are apparent at this level of resolution (,5% of the clones map either to other chromosomes or more than of the 143 clones in G-negative bands is higher than average (mean 0.55; x = 43, P , 0.005). These data confirm that dark G-bands are one band away from the expected position; Table 1). We found only eight locations where the cytogenetic data indicated that portions of more AT-rich than G-negative bands. The utility of a sequence-integrated cytogenetic resource is the sequence were misplaced within an earlier draft assembly (5 September 2000). The sequencing centres used these cytogenetic illustrated by two examples. In the first, clones are applied in findings to locate errors in the assembly and produce the later draft conventional FISH assays to rapidly narrow the search for candidate of improved quality (Table 2). genes disrupted or deregulated by translocations causing develop- FISH analyses of this clone collection reveal abundant paralogous mental disorders. The process is expedited by selection of clones relationships among sites dispersed across the human genome. Of assigned to the regions implicated by banding analyses. In a patient 1,243 clones randomly selected from the RPCI-11 library, 5.4% with multiple congenital malformations and mental retardation (DGAP012, http://dgap.harvard.edu), a breakpoint-spanning clone hybridize to more than one chromosomal location (see Supple- mentary Information 3). The entire collection includes 393 clones was identified (Fig. 1b). This clone spans a 170-kb interval contain- ing the gene for MKK7, a human mitogen-activated protein kinase, that together identify over 150 bands containing at least one segment with significant homology to one or more (up to 25) and a novel sequence with homology to the tre-2 oncogene, both plausible candidate genes. More typically, breakpoints will be other sites in the genome (see Supplementary Information 5). These data provide clues to duplications and exchanges that have occurred mapped to an interval between neighbouring clones. For example, within and between chromosomes. Among the 393 clones, 111 a translocation implicated in mental retardation in another patient contain blocks duplicated within the same chromosome; 282 maps to an interval containing at least 12 genes, including proto- hybridize to more than one chromosome. Paralogous relationships cadherin 8, a promising candidate given its exclusive expression in involving pericentromeric and subtelomeric regions of multiple fetal and adult brain . chromosomes are particularly frequent and complex. Clones in the In the second example, an array of around 2,000 BAC clones from collection also identify low-copy duplications specific to chromo- the collection is used to perform a genome-wide scan for segmental somes 1, 7, 11 and 16, the pseudoautosomal regions of X and Y, and aneuploidy by comparative genomic hybridization (CGH) (Fig. 3 and A. Snijders et al., in preparation). The array format offers better sites of the olfactory receptor gene family . Many previously 21,22 undescribed patterns were also observed; some were confirmed sensitivity and resolution than metaphase chromosomes, the traditional target for CGH , and, because the arrayed clones are with two or more clones, but others require further study to verify that they reflect true duplications. integrated into the draft, copy-number abnormalities can be related directly to sequence information. To illustrate the power of array Many of these duplications are functionally significant, as some have generated multigene families, and some are potential sites of CGH, the ML-2 cancer cell line was ‘karyotyped’ using the array. recombination events, which can result in chromosome abnormal- Array CGH revealed relative copy-number losses on 1p, 6q, 11q and ities. The cytogenetic data should greatly facilitate analyses of these 20p and gains of 12, 13 and 20q (Fig. 3). Copy-number abnorm- regions, which are likely to pose challenges to sequence assembly. alities on chromosomes 6, 11 and 20 were subsequently confirmed The sequence tags of 84% of the clones that hybridize to more than by FISH using clones predicted by array CGH to be included in the one site were placed in the 7 October 2000 draft assembly, and the region of loss. Several of these alterations were noted in previous location(s) were roughly consistent with at least one FISH observa- banding analyses (1p−,6q−, 11q−, +12, +13q+) , but array CGH tion for 88% of these clones. Collectively, the multisite clones locates the breakpoints precisely relative to BACs that reference specific locations in the sequence. highlight regions that are more likely to become entangled with other regions of the genome during sequence assembly than clones More than 7,500 clones now link the cytogenetic map and sequence of the human genome. Application of these reagents in with single FISH locations. Indeed, global BLAST analyses show that 956 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature 20q 1p 6q 11q 20p 1p Xq Position in genome 2.0 2.0 b c 1.5 1.5 1.0 1.0 0.5 0.5 0 0 0 1,000 2,000 3,000 4,000 5,000 6,000 0 10 20304050 60 70 80 90 Order on G3-radiation hybrid map Position on Genethon linkage map (cM) Figure 3 Copy-number analysis of myeloblastic leukaemia ML-2 cell line using CGH and a BAC normalized to the median ratio for all 2,000 clones on the array, ordered from 1pter genome-wide array of around 2,000 BAC clones. The ML-2 cell line has acquired to Xqter. Arrows, chromosomal regions showing significant copy number variations. The chromosomal abnormalities in addition to those present in the original tumour during lower ratio on the X indicates expected ratio for mismatched sex of test and reference long-term culture. CGH maps regions of abnormal copy number by comparing the relative DNAs. Fluorescence ratios of clones on chromosomes 11 (b) and 20 (c) are shown with efficiency with which test (Cy3-labelled ML-2 DNA) and reference (Cy5-labelled normal clones ordered according to position of their STSs on the G3 radiation hybrid or Genethon female DNA) hybridize to clones on the array. The array excludes clones that hybridize to linkage maps, respectively. multiple sites in the genome. a, Fluorescence ratios of Cy3 to Cy5 fluorescence for each 8. Cheung, V. G. et al. A resource of mapped human bacterial artificial chromosome clones. Genome Res. combination with increasingly detailed knowledge of genes and 9, 989 – 993 (1999). other functional motifs in the human sequence will transform the 9. Korenberg, J. R. et al. Human genome anatomy: BACs integrating the genetic and cytogenetic maps process of identifying genes that are altered in cancer and other for bridging genome and biomedicine. Genome Res 9, 994 – 1001 (1999). 10. Leversha, M. A., Dunham, I. & Carter, N. P. 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Nature Genet. 9, 347– 350 high resolution bivariate flow karyotyping. Am. J. Hum. Genet. 45, 738 – 752 (1989). (1995). 6. Mitelman, F. Catalog of Chromosome Aberrations in Cancer (Wiley, New York, 1998). 7. Eichler, E. E. Masquerading repeats: paralogous pitfalls of the human genome. Genome Res. 8, 758 – Supplementary information is available from Nature’s World-Wide Web site 762 (1998). (http://www.nature/com) or as paper copy from the London editorial office of Nature. NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 957 © 2001 Macmillan Magazines Ltd Ratio Ratio letters to nature Acknowledgements Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA; 6, Departments of Pediatrics and Human Genetics, Cedars-Sinai Medical Center, We thank M. Arcaro, M. Bakis, J. Burdick, J. Chang, H.-C. Chen, S. Chiu, Y. Fan, C. Harris, L. Haley, R. Hosseini, J. Kent, M. A. Leversha, J. Martin, L.-T. Nguyen, P. Quinn, Y. H. 8700 Beverly Boulevard, Los Angeles, California 90048, USA; 7, Computer Science Ramsey, T. Reppert, L. J. Rogers, J. Shreve, J. Stalica, M. Wang, T. Weber, A. M. Yavor, J. Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, Young, K. Zatloukal, and members of the TIGR BAC Ends Team for assistance. This work California 95064-1077, USA; 8, Department of Biology, California Institute of was supported by grants from NIH (NCI, NHGRI, NIDCD and NICHD), US DOE, NSF, Technology, Mail Code 147-75, Pasadena, California 91125, USA; 9, University of HHMI, PPG, Merck Genome Research Institute, Vysis, Inc., and start-up funds provided California San Francisco Cancer Center, Box 0808, San Francisco, California by Obstetrics and Gynecology at Brigham and Women’s Hospital. 94143-0808, USA; 10, Stanford University, Genome Lab, Mail Code 5120, Correspondence should be addressed to B.J.T. Stanford, California 94305-5120, USA; 11, Sanger Center, Wellcome Trust (e-mail: [email protected]). Genome Campus, Hinxton, Cambridge, CB10 1SA, UK; 12, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North C3-168, P.O. Box 19024, The BAC Resource Consortium Seattle, Washington 98109-1024, USA; 13, Department of Molecular 1 2 3 4 5 6 Biotechnology, University of Washington, Box 357730, Seattle, Washington 98195- V. G. Cheung *, N. Nowak *, W. Jang , I. R. Kirsch , S. Zhao , X.-N. Chen , 7 8 9 10 2 7730, USA; 14, Departments of Obstetrics and Gynecology and Pathology, T. S. Furey , U.-J. Kim †, W.-L. Kuo , M. Olivier , J. Conroy , 11 12 4 2 13 Brigham and Women’s Hospital, Amory Lab Building 3rd floor, Boston, A. Kasprzyk , H. Massa , R. Yonescu , S. Sait , C. Thoreen †, 9 14 15 1 11 Massachusetts 02115, USA; 15, Department of Human Genetics, Case Western A. Snijders , E. Lemyre , J. A. Bailey , A. Bruzel , W. D. Burrill , 11 13 11 12 16 Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA; S. M. Clegg , S. Collins , P. Dhami , C. Friedman ,C.S.Han , 14 8 14 17 1 16, Joint Genome Institute-Los Alamos National Laboratory, MS M888 B-N1, S. Herrick , J. Lee , A. H. Ligon , S. Lowry , M. Morley , 1 2,18 17 17 P.O. Box 1663, Los Alamos, New Mexico 87545, USA; 17, Joint Genome Institute- S. Narasimhan , K. Osoegawa , Z. Peng , I. Plajzer-Frick , 14 17 3 11 9 Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop 84-171, B. J. Quade , D. Scott , K. Sirotkin , A. A. Thorpe , J. W. Gray , 19 9 4 20 4 Berkeley, California 94720, USA; 18, Children’s Hospital Oakland Research J. Hudson , D. Pinkel , T. Ried , L. Rowen , G. L. Shen-Ong , 4 11 21 17 10 Institute, 747 52nd Street, Oakland, California 94609, USA; 19, Research R. L. Strausberg , E. Birney , D. F. Callen , J.-F. Cheng , D. R. Cox , 16 11 15 22 Genetics, 2130 Memorial Parkway, Huntsville, Alabama 35801, USA; N. A. Doggett , N. P. Carter , E. E. Eichler , D. Haussler , 6 14 9 3 20, Institute for Systems Biology, 4225 Roosevelt Way NE, Suite 200, Seattle, J. R. Korenberg , C. C. Morton , D. Albertson , G. Schuler ,P. J. de 2,18 12 Washington 98105-6099, USA; 21, Department of Cytogenetics and Molecular Jong & B. J. Trask Genetics, Women’s and Children’s Hospital, 72 King William Road, * These authors contributed equally to this work. North Adelaide, South Australia 5006, Australia; 22, Howard Hughes Medical 1, Department of Pediatrics, University of Pennsylvania, The Children’s Hospital Institute, Computer Science Department, University of California Santa Cruz, of Philadelphia, 3516 Civic Center Boulevard, ARC 516, Philadelphia, 1156 High Street, Santa Cruz, California 95064– 1077, USA Pennsylvania 19104, USA; 2, Roswell Park Cancer Institute, Elm and Carleton Present addresses: PanGenomics, 6401 Foothill Boulevard, Tujunga, Street, Buffalo, New York 14263, USA; 3, National Center for Biotechnology California 91024, USA (U.-J.K.); Harvard Medical School, 240 Longwood Information, National Library of Medicine, Building 38A/Room 8N805, Avenue, Cell Biology, Cambridge, Massachusetts 02115, USA (C.T.); Gene Logic, Bethesda, Maryland 20894, USA; 4, National Cancer Institute, NIH, Building 10/ Inc., 708 Quince Orchard Road, Gaithersburg, Maryland 20878, USA Room 12N214, Bethesda, Maryland 20889-5105, USA; 5, The Institute for (G.L.S.-O.). 958 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd

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Published: Feb 15, 2001

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