Our sequence-tagged site-content map of chromosome 12 is now integrated with the whole-genome fingerprinting effort. It provides accurate and nearly complete bacterial clone coverage of chromosome 12. We propose that this integrated mapping protocol serves as a model for constructing physical maps for entire genomes

Chiu, Denise

Decker, Jeremy

Emerling, Suzanne

Gibbs, Richard

Han, Jinghua

Ioshikhes, Ilya

Kim, Rachel

Kirsch, Ilan

Kucherlapati, Raju

Lau, Stephanie

Lee, Eunice

Lenz, Jack

Marondel, Ivonne

McPherson, John

Miller, Ashley

Montgomery, Kate

Renault, Beatrice

Ried, Thomas

Scherer, Steven

Sekhon, Mandeep

Shim, Cecilia

Song, Kyuyoung

Vundavalli, Murty V.

Yonescu, Raluca

Yoon, Sung-Joo Kim

Columbia University Academic Commons

letters to natureNATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 945was extensively cross-checked against the radiation hybrid map andwas further reinforced by restriction fingerprinting of all mappedBACs15.The greatest challenges were posed by massive, NRY-specificamplified regions (or amplicons), which comprise about one-third of the euchromatic NRY. Of the 758 STSs on which the mapis built, 136 are present at two or more locations in the NRY.Although we avoided such repetitive STSs in favour of single-copySTSs wherever possible, substantial portions of the euchromaticNRY contained little or no single-copy sequence. For many suchamplicons, BACs derived from different copies could not bedistinguished by STS content or restriction fingerprinting15. Inmany cases, we distinguished among amplicon copies (and theBACs corresponding to them) by typing ‘sequence family variants’(SFVs)5. SFVs are subtle differences (for example, single-nucleotidesubstitutions or dinucleotide repeat length alterations) betweenclosely related but non-allelic sequences. We were analysing BACsfrom only one male’s Y chromosome, so these subtle sequencedifferences could not represent allelic variants. In general, weidentified SFVs only after comparing the DNA sequences of BACsthat originated from distinct copies, despite having similar STScontent. Thus, mapping and sequencing were inseparable, iterativeactivities in amplicon-rich regions.The euchromatic NRYamplicons are diverse in composition, size,copy number and orientation (Fig. 1), with some occurring astandem repeats, others as inverted repeats, and still others dispersedthroughout both arms of the chromosome. The euchromaticamplicons are well populated with testis-specific gene familiesthat may be critical in spermatogenesis (see Fig. 1 in SupplementaryInformation)1,2.One pair of amplicons is of particular interest in the context ofhuman variation. Highlighted in Fig. 1 (arrows) are two units, each300 kb long, that exist in opposite orientations on the short arm.These inverted repeats bound a region of around 3.5 Mb that occursin one orientation (Fig. 1 in Supplementary Information) in themale from whom the BAC library was constructed, but in theopposite orientation in the existing map of naturally occurringdeletions6. This may reflect variation among men for a 3.5-Mbinversion1,16, perhaps the result of homologous recombinationbetween the 300-kb inverted repeats flanking the inverted segment.Large Y-chromosome inversions are postulated to have been crucialin the evolution of the human sex chromosomes12, and this 3.5-Mbinversion may be one of many massive NRY variants that exist inmodern populations. MReceived 16 November; accepted 21 December 2000.1. Vogt, P. H. et al. Report of the Third International Workshop on Y Chromosome Mapping 1997.Cytogenet. Cell Genet. 79, 1–20 (1997).2. Lahn, B. T. & Page, D. C. Functional coherence of the human Y chromosome. Science 278, 675–680(1997).3. Foote, S., Vollrath, D., Hilton, A. & Page, D. C. The human Y chromosome: Overlapping DNA clonesspanning the euchromatic region. Science 258, 60–66 (1992).4. Reijo, R. et al. Diverse spermatogenic defects in humans caused by Y chromosome deletionsencompassing a novel RNA-binding protein gene. Nature Genet. 10, 383–393 (1995).5. Saxena, R. et al. Four DAZ genes in two clusters found in the AZFc region of the human Ychromosome. Genomics 67, 256–267 (2000).6. Vollrath, D. et al. The human Y chromosome: A 43-interval map based on naturally occurringdeletions. Science 258, 52–59 (1992).7. Hudson, T. J. et al. An STS-based map of the human genome. Science 270, 1945–1954 (1995).8. Gyapay, G. et al. A radiation hybrid map of the human genome. Hum. Mol. Genet. 5, 339–346 (1996).9. Stewart, E. A. et al. An STS-based radiation hybrid map of the human genome. Genome Res. 7, 422–433 (1997).10. Deloukas, P. et al. A physical map of 30,000 human genes. Science 282, 744–746 (1998).11. Lunetta, K. L., Boehnke, M., Lange, K. & Cox, D. R. Selected locus and multiple panel models forradiation hybrid mapping. Am. J. Hum. Genet. 59, 717–725 (1996).12. Lahn, B. T. & Page, D. C. Four evolutionary strata on the human X chromosome. Science 286, 964–967(1999).13. Mumm, S., Molini, B., Terrell, J., Srivastava, A. & Schlessinger, D. Evolutionary features of the 4-MbXq21.3 XY homology region revealed by a map at 60-kb resolution. Genome Res. 7, 307–314 (1997).14. Schwartz, A. et al. Reconstructing hominid Y evolution: X-homologous block, created by X-Ytransposition, was disrupted by Yp inversion through LINE-LINE recombination. Hum. Mol. Genet.7, 1–11 (1998).15. The International Human Genome Mapping Consortium. A physical map of the human genome.Nature 409, 934–941 (2001).16. Jobling, M. A. et al. A selective difference between human Y-chromosomal DNA haplotypes. Curr.Biol. 8, 1391–1394 (1998).Supplementary information is available on Nature’s World-Wide Web site(http://www.nature.com) or as paper copy from the London editorial office of Nature.AcknowledgementsWe thank C. Nusbaum and T. Hudson for materials and advice on radiation hybridmapping; J. Crockett, J. Fedele, N. Florence, H. Grover, C. McCabe, N. Mudd, S. Sasso,D. Scheer, R. Seim and P. Shelby for technical contributions; and D. Berry, J. Bradley,Y. Lim, A. Lin, D. Menke, M. Royce-Tolland, J. Saionz and J. Wang for comments on themanuscript. Supported in part by NIH.Correspondence and requests for materials should be addressed to D.C.P.(e-mail: dcpage@wi.mit.edu)..................................................................A high-resolution map of humanchromosome 12Kate T. Montgomery*, Eunice Lee*, Ashley Miller*, Stephanie Lau*,Cecilia Shim*, Jeremy Decker*, Denise Chiu*, Suzanne Emerling*,Mandeep Sekhon†, Rachel Kim*, Jack Lenz*, Jinghua Han*,Ilya Ioshikhes*, Beatrice Renault*, Ivonne Marondel*,Sung-Joo Kim Yoon‡, Kyuyoung Song§, V. V. V. S. Murtyk,Steven Scherer¶, Raluca Yonescu#, Ilan R. Kirsch#, Thomas Ried#,John McPherson†, Richard Gibbs¶ & Raju Kucherlapati**Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx,New York 10461, USA‡ Catholic University Medical College Research Institutes of Medical Sciences,Seoul 137-404, Korea§ University of Ulsan College of Medicine, Seoul 138-736, KoreakDepartment of Pathology, Columbia University College of Physicians andSurgeons, New York, New York 10032, USA¶ Department of Molecular and Human Genetics, Baylor College of Medicine,Houston, Texas 77030, USA# Genetics Department, Medicine Branch, National Cancer Institute, NationalInstitutes of Health, Bethesda, Maryland 20892, USA† Genome Sequencing Center, Washington University School of Medicine,St. Louis, Missouri 63108, USA................. ......................... ......................... ......................... ......................... .........................Our sequence-tagged site-content map of chromosome 12 is nowintegrated with the whole-genome fingerprinting effort1,2. Itprovides accurate and nearly complete bacterial clone coverageof chromosome 12. We propose that this integrated mappingprotocol serves as a model for constructing physical maps forentire genomes.Human chromosome 12 has been estimated to be 125 megabases(Mb) in size. Several low-density clone maps, genetic linkage mapsand radiation hybrid maps that include chromosome 12 have beendescribed3–5. The map presented here is based on large bacterialclones that include bacterial artificial chromosomes (BACs), P1(phage) artificial chromosomes (PACs; http://www.chori.org/bacpac) and a few cosmids6. To construct the map, we divided thechromosome into 1–2 centiMorgan (cM) intervals and assembledall sequence-tagged site (STS) markers known to be in each interval.We used probes derived from the non-polymorphic markers in aninterval to screen colony arrays representing six to ten genomeequivalents from the RPCI libraries. To extend contigs and linkadjacent contigs, we used STSs corresponding to end sequences ofBACs at the outer boundaries of each contig to screen members ofthe contig from which they were derived as well as possible adjacentcontigs. This process was reiterated until the region was covered by© 2001 Macmillan Magazines Ltdletters to nature946 NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.comoverlapping clones7. The STS-content map was assembled manuallyand the data stored in a relational database. A representative portionof the map corresponding to 5 cM of 12p can be seen in Supple-mentary Information; the entire map is available on our website(http://sequence.aecom. yu.edu/chr12/).Our map contains 3,090 STS markers. Of these, 438 are poly-morphic, 1,836 are non-polymorphic and the remaining 816 arebased on genes or expressed sequence tags (ESTs). These markerswere obtained from gene maps8, linkage maps3,9,10 or the WhiteheadInstitute radiation hybrid map (http://www.genome.wi.mit.edu),or developed at the Albert Einstein College of Medicine GenomeCenter. If the size of chromosome 12 is 125 Mb, the STS markersprovide an average resolution of 40 kb. There are more than 5,300large-insert clones on the map, and each marker is present in anaverage of 8.1 bacterial clones. This depth provides a high level ofconfidence in the quality of the map. Originally, 1,154 tiling pathand seeder clones were identified for sequencing chromosome 12;1,115 of them are being sequenced. The map indicates that some ofthe seeder clones are redundant and do not need to be finished. Theestimated minimal set of clones covering chromosome 12 is 1,025.On average, adjacent clones overlap by 30%. As the average size ofthe clones in the RPCI-11 library is 174 kb, we can estimate thephysical size of chromosome 12 excluding the gaps (see Supple-mentary Information) to be 125 Mb.We assessed the accuracy of the map in two ways. First, weanalysed tiling path clones with any available sequence to deter-mine whether they overlapped as predicted by the map. Usingelectronic polymerase chain reaction (ePCR), BLAST (http://www.ncbi.nlm.nih.gov/) or the annotation of finished clones,most overlaps were confirmed. Second, we analysed clones locatedat 1-Mb intervals along the chromosome using fluorescence in situhybridization (FISH) as part of the Cancer Chromosome Aberra-tion Project (CCAP)11,12. We chose 120 clones, and their orderproved to be consistent with the order in the integrated map. Thenames of the clones, their cytogenetic locations, and details of theirdistribution are available at http://www.ncbi.nlm.nih.gov/CCAP/.The STS-content map of chromosome 12 has been compared andintegrated with the whole-genome map generated by BAC clonefingerprinting. The RPCI-11 BAC library was the primary resourcefor both maps, so the two are fully compatible. The comparisonvalidated both methods and revealed them to be complementary.When the comparison was initiated, the STS-content map con-tained 74 contigs anchored to the genetic map. The whole genomemap contained 75 contigs anchored to the G4 radiation hybrid map4and nine additional contigs that were assigned to chromosome 12but unanchored.The integrated map now consists of 18 contigs that are not linkedby PCR or sequence data (see Supplementary Information). Thegenetic markers on the STS map provide strong evidence for theorientation and order of most of these 18 contigs. This permits thedirect comparison of fingerprints of end clones that are adjacent toeach other on the map. In two cases we find that the fingerprintsoverlap and consider these contigs to be manually closed2; in onecase we know by sequence that the clones do not overlap and in onethe fingerprints are difficult to interpret (see below). The remaining12 pairs of end clones do not have similar fingerprints and addi-tional clones may be required to achieve contiguity. By thesecriteria, we consider the chromosome 12 map to have 14 gaps(see Supplementary Information), excluding the centromere, in itsbacterial clone coverage.We previously reported a yeast artificial chromosome (YAC) mapof chromosome 12 (ref. 5). When the YAC, BAC and whole-genomefingerprint maps are combined, YACs cover all but five of the gaps,so we expect that these gaps are less than 1 Mb. The gaps not coveredby YACs include one in a repetitive region at 43.6 cM (describedbelow), and four between 137.5 and 169.1 cM.Some of the gaps differ, and may reflect significant genomicfeatures. Understanding these regions will benefit the mapping andsequencing of this and other genomes. For example, the gap at13.9 cM is covered by a YAC, but we have been unable to identify abacterial clone to fill it, despite screening the libraries RPCI-1, 3, 4,5, 11 and 13 several times with different probes. Furthermore,sequence is currently available for most of the clones in the region,but there are no sequence hits in any of the databases for the ends ofthe gap. This is the only region of the chromosome that appears tobe missing from the available bacterial clone libraries, suggestingthat true deficiencies in the bacterial libraries may be rare.Two gaps appear in the region corresponding to 43.6 cM on 12p.Screening of the 12X BAC library with markers in this region yieldeda set of clones whose number far exceeded unique representation ofthis region in the genome. STS-content analysis of the clonesshowed that each marker was positive for as many as 30–40clones and the order of the clones could not be determined logically.These clones had similar restriction patterns and fell into very densefingerprint contigs that included clones containing markers onchromosomes 2, 3, 11, 12 and 14. The combined fingerprint andPCR results indicate that this region may contain large duplicationsreminiscent of those described on chromosome 22 (refs 13,14) andit may also represent a region duplicated in other parts of thegenome. The fingerprints of the clones flanking one gap in thisregion have been examined and the data strongly support coverage.Neither fingerprint nor STS data can currently resolve the secondgap. This repetitive region may represent one of the most difficulttypes of region to map and sequence in full, in both the human andother genomes.This map is one of the most complete human chromosome mapsavailable. The known marker order for each clone allows easyassembly of draft sequence and the map will be invaluable indirecting efforts towards completing the sequencing of chromo-some 12 as well as future functional genomic studies. MReceived 16 November; accepted 21 December 2000.1. Marra, M. A. et al. High throughput fingerprint analysis of large-insert clones. Genome Res. 11, 1072–1084 (1997).2. The International Human Genome Mapping Consortium. A physical map of the human genome.Nature 409, 934–941 (2001).3. Dib, C. et al. A comprehensive genetic map of the human genome based on 5,264 microsatellites.Nature 380, 152–154 (1996).4. Stewart, E. A. et al. An STS-based radiation hybrid map of the human genome. Genome Res. 7, 422–433 (1997).5. Krauter, K. et al. A second generation YAC contig map of human chromosome 12. Nature 377(Suppl.), 321–334 (1995).6. Montgomery, K. T. et al. Characterization of two chromosome 12 cosmid libraries and developmentof STSs from cosmids mapped by FISH. Genomics 17, 682–693 (1993).7. Renault, B. et al. A sequence-ready physical map of a region of 12q24. 1. Genomics 45, 271–278(1997).8. Deloukas, P. et al. A physical map of 30,000 human genes. Science 282, 744–746 (1998).9. Murray, J. C. et al. A comprehensive human linkage map with centimorgan density. Science 265, 2049–2054 (1994).10. Broman, K. W., Murray, J. C., Sheffield, V. C., White, R. L. & Weber, J. L. Comprehensive humangenetic maps: individual and sex-specific variation in recombination. Am. J. Hum. Genet. 63, 861–869(1998).11. Kirsch, I. R. et al. A systematic, high-resolution linkage of the cytogenetic and physical maps of thehuman genome. Nature Genet. 24, 339–340 (2000).12. Ried, T., Baldini, A., Rand, T. C. & Ward, D. C. Simultaneous visualization of seven different DNAprobes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.Proc. Natl Acad. Sci. USA 89, 1388–1392 (1992).13. Edelmann, L. et al. A common molecular basis for rearrangement disorders on chromosome 22q11.Hum. Mol. Genet. 8, 1157–1167 (1999).14. Dunham, I. et al. The DNA sequence of human chromosome 22. Nature. 402, 489–495 (1999).Supplementary information is available from Nature’s World-Wide Web site(http://www.nature.com) or as paper copy from the London editorial office of Nature.AcknowledgementsThis work is supported by the NIH and the Human Genetics program at Einstein. TheCancer Chromosome Aberrations Project (CCAP) is funded through the office of thedirector, NCI. We appreciate comments on the manuscript from F. Collins and theassistance of J. Goltz. This work was inspired by F. Ruddle.Correspondence and requests for materials should be addressed to R.K.(e-mail: kucherla@aecom.yu.edu).© 2001 Macmillan Magazines Ltd

A high-resolution map of human chromosome 12

A high-resolution map of human chromosome 12

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