The genome sequence of Drosophila melanogaster

Science

Volumn 287, Issue 5461, 2000, Pages 2185-2195

  Adams, M D ^a   Celniker, S E ^b   Holt, R A ^a   Evans, C A ^a   Gocayne, J D ^a   Amanatides, P G ^a   Scherer, S E ^c   Li, P W ^a   Hoskins, R A ^b   Galle, R F ^b   George, R A ^b   Lewis, S E ^d   Richards, S ^b   Ashburner, M ^e   Henderson, S N ^a   Sutton, G G ^a   Wortman, J R ^a   Yandell, M D ^a   Zhang, Q ^a   Chen, L X ^a   Brandon, R C ^a   Rogers, Y H C ^a   Blazej, R G ^b   Champe, M ^b   Pfeiffer, B D ^b   Wan, K H ^b   Doyle, C ^b   Baxter, E G ^b   Helt, G ^f   Nelson, C R ^d   Gabor Miklos, G L ^g   Abril, J F ^h   Agbayani, A ^b   An, H J ^a   Andrews Pfannkoch, C ^a   Baldwin, D ^a   Ballew, R M ^a   Basu, A ^a   Baxendale, J ^a   Bayraktaroglu, L ⁱ   Beasley, E M ^a   Beeson, K Y ^a   Benos, P V ^j   Berman, B P ^b   Bhandari, D ^a   Bolshakov, S ^k   Borkova, D ^l   Botchan, M R ^d   Bouck, J ^c   Brokstein, P ^d   Brottier, P ^m   Burtis, K C ⁿ   Busam, D A ^a   Butler, H ^o   Cadieu, E ^p   Center, A ^a   Chandra, I ^a   Michael Cherry, J ^q   Cawley, S ^d   Dahlke, C ^a   Davenport, L B ^a   Davies, P ^a   de Pablos, B ^r   Delcher, A ^a   Deng, Z ^a   Deslattes Mays, A ^a   Dew, I ^a   Dietz, S M ^a   Dodson, K ^a   Doup, L E ^a   Downes, M ^s   Dugan Rocha, S ^c   Dunkov, B C ^t   Dunn, P ^a   Durbin, K J ^c   Evangelista, C C ^a   Ferraz, C ^u   Ferriera, S ^a   Fleischmann, W ^e   Fosler, C ^a   Gabrielian, A E ^a   Garg, N S ^a   Gelbart, W M ⁱ   Glasser, K ^a   Glodek, A ^a   Gong, F ^a   Harley Gorrell, J ^c   Gu, Z ^a   Guan, P ^a   Harris, M ^a   Harris, N L ^b   Harvey, D ^d   Heiman, T J ^a   Hernandez, J R ^c   Houck, J ^a   Hostin, D ^a   Houston, K A ^b   Howland, T J ^a   Wei, M H ^a   Ibegwam, C ^a   Jalali, M ^a   Kalush, F ^a   Karpen, G H ^s   Ke, Z ^a   Kennison, J A ^v   Ketchum, K A ^a   Kimmel, B E ^b   Kodira, C D ^a   Kraft, C ^a   Kravitz, S ^a   Kulp, D ^f   Lai, Z ^a   Lasko, P ^w   Lei, Y ^a   Levitsky, A A ^a   Li, J ^a   Li, Z ^a   Liang, Y ^a   Lin, X ^x   Liu, X ^a   Mattei, B ^a   McIntosh, T C ^a   McLeod, M P ^c   McPherson, D ^a   Merkulov, G ^a   Milshina, N V ^a   Mobarry, C ^a   Morris, J ^f   Moshrefi, A ^b   Mount, S M ^y   Moy, M ^a   Murphy, B ^a   Murphy, L ^e   Muzny, D M ^c   Nelson, D L ^c   Nelson, D R ^z   Nelson, K A ^a   Nixon, K ^b   Nusskern, D R ^a   Pacleb, J M ^b   Palazzolo, M ^b   Pittman, G S ^a   Pan, S ^a   Pollard, J ^a   Puri, V ^a   Reese, M G ^d   Reinert, K ^a   Remington, K ^a   Saunders, R D C ^aa,ab   Scheeler, F ^a   Shen, H ^c   Christopher Shue, B ^a   Siden Kiamos, I ^k   Simpson, M ^a   Skupski, M P ^a   Smith, T ^a   Spier, E ^a   Spradling, A C ^ac   Stapleton, M ^b   Strong, R ^a   Sun, E ^a   Svirskas, R ^ad   Tector, C ^a   Turner, R ^a   Venter, E ^a   Wang, A H ^a   Wang, X ^a   Wang, Z Y ^a   Wassarman, D A ^v   Weinstock, G M ^c   Weissenbach, J ^m   Williams, S M ^a   Woodage, T ^a   Worley, K C ^c   Wu, D ^a   Yang, S ^b   Alison Yao, Q ^a   Ye, J ^a   Yeh, R F ^d   Zaveri, J S ^a   Zhan, M ^a   Zhang, G ^a   Zhao, Q ^a   Zheng, L ^a   Zheng, X H ^a   Zhong, F N ^a   Zhong, W ^a   Zhou, X ^c   Zhu, S ^a   Zhu, X ^a   Smith, H O ^a   Gibbs, R A ^c   Myers, E W ^a   Rubin, G M ^d   Craig Venter, J ^a   more..

^a CELERA GENOMICS   (United States)

^b LAWRENCE BERKELEY NATIONAL LABORATORY   (United States)

^c BAYLOR COLLEGE OF MEDICINE   (United States)

^d UNIVERSITY OF CALIFORNIA   (United States)

^e WELLCOME TRUST SANGER INSTITUTE   (United Kingdom)

^f Neomorphic Inc   (United States)

^g GenetixXpress Proprietary Limited   (Australia)

^h INST MUNIC D INVESTIGACIO MEDICA   (Spain)

ⁱ HARVARD UNIVERSITY   (United States)

^j WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^k INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY   (Greece)

^l EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

^m CENTRE NATIONAL DE SÉQUENÇAGE   (France)

ⁿ UNIVERSITY OF CALIFORNIA   (United States)

^o UNIVERSITY OF CAMBRIDGE   (United Kingdom)

^p Institute of Electronics and Telecommunicalions of Rennes   (France)

^q STANFORD UNIVERSITY   (United States)

^r UNIVERSIDAD AUTÓNOMA DE MADRID   (Spain)

^s SALK INSTITUTE FOR BIOLOGICAL STUDIES   (United States)

^t University of Arizona   (United States)

^u UNIV MONTPELLIER   (France)

^v EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT   (United States)

^w MCGILL UNIVERSITY   (Canada)

^x J CRAIG VENTER INSTITUTE   (United States)

^y Bldg 142   (United States)

^z UNIVERSITY OF TENNESSEE HEALTH SCIENCE CENTER   (United States)

^aa UNIVERSITY OF DUNDEE   (United Kingdom)

^ab OPEN UNIVERSITY   (United Kingdom)

^ac GEOPHYSICAL LABORATORY   (United States)

^ad MOTOROLA INC   (United States)

more..

Author keywords

[No Author keywords available]

Indexed keywords

DROSOPHILA MELANOGASTER; EUCHROMATIN; EUKARYOTE; GENE STRUCTURE; GENOME; HETEROCHROMATIN; MOLECULAR CLONING; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; REVIEW; SEQUENCE ANALYSIS;

BACTERIA (MICROORGANISMS); CAENORHABDITIS ELEGANS; DROSOPHILA MELANOGASTER; EUKARYOTA; INVERTEBRATA; MELANOGASTER;

EID: 0034708480     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5461.2185     Document Type: Review

References (91)

1. G. L. G. Miklos and G. M. Rubin, Cell 86, 521 (1996).
2. A. S. Spradling et al., Genetics 153, 135 (1999).
3. M. Ashburner et al., Genetics 153, 179 (1999).
4. J. C. Venter et al., Science 280, 1540 (1998).
5. G. M. Rubin and E. Lewis, Science 287, 2216 (2000).
6. D. L. Hartl et al., Trends Genet. 8, 70 (1992).
7. R. D. Fleischmann et al., Science 269, 496 (1995); C. M. Fraser and R. D. Fleischmann, Electrophoresis 18, 1207 (1997).
8. R. D. Fleischmann et al., Science 269, 496 (1995); C. M. Fraser and R. D. Fleischmann, Electrophoresis 18, 1207 (1997).
9. J. L. Weber and E. W. Myers, Genome Res. 7, 409 (1997).
10. J. C. Venter, H. O. Smith, L. Hood, Nature 381, 364 (1996).
11. R. Hoskins et al., Science 287, 2271 (2000).
12. E. W. Myers et al., Science 287, 2196 (2000).
13. A number of methods were used to close gaps. Whenever possible, gaps were localized to a chromosome region and a spanning genomic clone was identified. When a spanning clone could be identified, it was used as a template for sequencing. The sequencing approach was determined by the gap size. For gaps smaller than 1 kb, BAC templates were sequenced directly with custom primers. For gaps larger than 1 kb, 3-kb plasmids or M13 clones from the clone-based draft sequencing were sequenced by directed methods, or 10-kb plasmids from the WGS sequencing project were sequenced by random transposon-based methods. If no 3-kb or 10-kb plasmid could be identified, PCR products were amplified from BAC clones or genomic DNA and end-sequenced directly with the PCR primers.
14. K. S. Weiler and B. T. Wakimoto, Annu. Rev. Genet. 29, 577 (1995); S. Henikoff, Biochem. Biophys. Acta 1470, 1 (2000); S. Pimpinelli et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3804 (1995); A. R. Lohe, A. J. Hilliker, P. A. Roberts, Genetics 134, 1149 (1993).
15. K. S. Weiler and B. T. Wakimoto, Annu. Rev. Genet. 29, 577 (1995); S. Henikoff, Biochem. Biophys. Acta 1470, 1 (2000); S. Pimpinelli et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3804 (1995); A. R. Lohe, A. J. Hilliker, P. A. Roberts, Genetics 134, 1149 (1993).
16. K. S. Weiler and B. T. Wakimoto, Annu. Rev. Genet. 29, 577 (1995); S. Henikoff, Biochem. Biophys. Acta 1470, 1 (2000); S. Pimpinelli et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3804 (1995); A. R. Lohe, A. J. Hilliker, P. A. Roberts, Genetics 134, 1149 (1993).
17. K. S. Weiler and B. T. Wakimoto, Annu. Rev. Genet. 29, 577 (1995); S. Henikoff, Biochem. Biophys. Acta 1470, 1 (2000); S. Pimpinelli et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3804 (1995); A. R. Lohe, A. J. Hilliker, P. A. Roberts, Genetics 134, 1149 (1993).
18. G. L. G. Miklos, M. Yamamoto, J. Davies, V. Pirrotta, Proc. Natl. Acad. Sci U.S.A. 85, 2051 (1988).
19. See ftp.ebi.ac.uk/pub/databases/edgp/sequence_sets/ nuclear_cds_set.embl.v2.9.Z.
20. The genes found in unscaffolded sequence were Su(Ste) (FlyBase identifier FBgn0003582) on the Y chromosome, His1 (FBgn0001195) and His4 (FBgn0001200) (histone genes were screened out before assembly), rbp13 (FBgn0014016), and idr (FBgn0020850).
21. C. Burge and S. Karlin, J. Mol. Biol. 268, 78 (1997).
22. M. G. Reese, D. Kulp, H. Tammana, D. Haussler, Genome Res., in press.
23. -4 were then processed on the basis of their high-scoring pair (HSP) coordinates on the contig to remove redundant hits, retaining hits that supported possible alternative splicing. This procedure was performed separately by hits to particular organisms so as not to exclude HSPs that support the same gene structure. Sequences producing BLAST hits judged to be informative, nonredundant, and sufficiently similar to the contig sequence were then realigned to the contig with Sim4 [L. Florea, G. Hartzell, Z. Zhang, G. M. Rubin, W. Miller, Genome Res. 8, 967 (1998)] for ESTs, and with Lap [X. Huang, M. D. Adams, H. Zhou, A. R. Kerlavage, Genomics 46, 37 (1995)] for proteins. Because both of these algorithms take splicing into account, the resulting alignments usually respect intron-exon boundaries and thus facilitate human annotation. Some regions of the genome may be underannotated because the bulk of the annotation work was done on an earlier assembly version. Continued updates will be available through FlyBase.
24. -4 were then processed on the basis of their high-scoring pair (HSP) coordinates on the contig to remove redundant hits, retaining hits that supported possible alternative splicing. This procedure was performed separately by hits to particular organisms so as not to exclude HSPs that support the same gene structure. Sequences producing BLAST hits judged to be informative, nonredundant, and sufficiently similar to the contig sequence were then realigned to the contig with Sim4 [L. Florea, G. Hartzell, Z. Zhang, G. M. Rubin, W. Miller, Genome Res. 8, 967 (1998)] for ESTs, and with Lap [X. Huang, M. D. Adams, H. Zhou, A. R. Kerlavage, Genomics 46, 37 (1995)] for proteins. Because both of these algorithms take splicing into account, the resulting alignments usually respect intron-exon boundaries and thus facilitate human annotation. Some regions of the genome may be underannotated because the bulk of the annotation work was done on an earlier assembly version. Continued updates will be available through FlyBase.
25. M. G. Reese, G. Hartzell, N. L. Harris, U. Ohler, S. E. Lewis, Genome Res., in press.
26. G. M. Rubin et al., Science 287, 2222 (2000).
27. See the Gene Ontology Web site (www.geneontology. org).
28. See the Saccharomyces Genome Database Web site (http://genome-www.stanford.edu/Saccharomyces).
29. D. Allen and J. Blake, Mouse Genome Informatics (www.informatics.jax.org).
30. S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997).
31. S. M. Mount et al., Nucleic Acids Res. 20, 4255 (1992).
32. The C. elegans Sequencing Consortium, Science 282, 2012 (1998).
33. X. Lin et al., Nature 402, 761 (1999).
34. G. M. Rubin et al., Science 287, 2204 (2000).
35. A. Dutta and S. P. Bell, Annu. Rev. Cell Dev. Biol. 13, 293 (1997).
36. I. Chesnokov, M. Gossen, D. Remus, M. Botchan, Genes Dev. 13, 1288 (1999).
37. G. Feger, Gene 227, 149 (1999).
38. D. T. Pak et al., Cell 97, 311 (1997); J. Rohrbough, S. Pinto, R. M. Mihalek, T. Tully, K. Broadie, Neuron 23, 55 (1999).
39. D. T. Pak et al., Cell 97, 311 (1997); J. Rohrbough, S. Pinto, R. M. Mihalek, T. Tully, K. Broadie, Neuron 23, 55 (1999).
40. S. Waga, G. J. Hannon, D. Beach, B. Stillman, Nature 369, 574 (1994); H. Flores-Rozas et al., Proc. Natl. Acad. Sci. U.S.A. 91, 8655 (1994).
41. S. Waga, G. J. Hannon, D. Beach, B. Stillman, Nature 369, 574 (1994); H. Flores-Rozas et al., Proc. Natl. Acad. Sci. U.S.A. 91, 8655 (1994).
42. R. Jessberger, C. Frei, S. M. Gasser, Curr. Opin. Genet. Dev. 8, 254 (1998); T. Hirano, Curr. Opin. Genet. Dev. 10, 317 (1998); A. V. Strunnikov, Trends Cell Biol. 8, 454 (1998).
43. R. Jessberger, C. Frei, S. M. Gasser, Curr. Opin. Genet. Dev. 8, 254 (1998); T. Hirano, Curr. Opin. Genet. Dev. 10, 317 (1998); A. V. Strunnikov, Trends Cell Biol. 8, 454 (1998).
44. R. Jessberger, C. Frei, S. M. Gasser, Curr. Opin. Genet. Dev. 8, 254 (1998); T. Hirano, Curr. Opin. Genet. Dev. 10, 317 (1998); A. V. Strunnikov, Trends Cell Biol. 8, 454 (1998).
45. R. Saffery et al., Hum. Mol. Genet. 9, 175 (2000); J. M. Craig, W. C. Earnshaw, P. Vagnarelli, Exp. Cell Res. 246, 249 (1999); R. Saffery et al., Chromosome Res. 7, 261 (1996).
46. R. Saffery et al., Hum. Mol. Genet. 9, 175 (2000); J. M. Craig, W. C. Earnshaw, P. Vagnarelli, Exp. Cell Res. 246, 249 (1999); R. Saffery et al., Chromosome Res. 7, 261 (1996).
47. R. Saffery et al., Hum. Mol. Genet. 9, 175 (2000); J. M. Craig, W. C. Earnshaw, P. Vagnarelli, Exp. Cell Res. 246, 249 (1999); R. Saffery et al., Chromosome Res. 7, 261 (1996).
48. R. Belotserkovskaya and S. L. Berger, Crit. Rev. Eukaryotic Gene Expr. 9, 221 (1999).
49. J. A. Eisen, K. S. Sweder, P. C. Hanawalt, Nucleic Acids Res. 23, 2715 (1995); K. J. Pollard and C. L. Peterson, Bioessays 20, 771 (1998).
50. J. A. Eisen, K. S. Sweder, P. C. Hanawalt, Nucleic Acids Res. 23, 2715 (1995); K. J. Pollard and C. L. Peterson, Bioessays 20, 771 (1998).
51. E. V. Koonin, S. Zhou, J. C. Lucchesi, Nucleic Acids Res. 23, 4229 (1995).
52. F. Jeanmougin et al., Trends Biochem. Sci. 22, 151 (1997); F. Winston and C. D. Allis, Nature Struct. Biol. 6, 601 (1999).
53. F. Jeanmougin et al., Trends Biochem. Sci. 22, 151 (1997); F. Winston and C. D. Allis, Nature Struct. Biol. 6, 601 (1999).
54. R. W. Levis, Mol. Gen. Genet. 236, 440 (1993); H. Biessmann and J. M. Mason, Chromosoma 106, 63 (1997).
55. R. W. Levis, Mol. Gen. Genet. 236, 440 (1993); H. Biessmann and J. M. Mason, Chromosoma 106, 63 (1997).
56. P. Gallinari and J. Jiricny, Nature 383, 735 (1996).
57. B. Flores and W. Engels, Proc. Natl. Acad. Sci. U.S.A. 96, 2964 (1999).
58. K. Kusano, M. E. Berres, W. R. Engels, Genetics 151, 1027 (1999); J. J. Sekelsky, M. H. Brodsky, G. M. Rubin, R. S. Hawley, Nucleic Acids Res. 27, 3762 (1999).
59. K. Kusano, M. E. Berres, W. R. Engels, Genetics 151, 1027 (1999); J. J. Sekelsky, M. H. Brodsky, G. M. Rubin, R. S. Hawley, Nucleic Acids Res. 27, 3762 (1999).
60. M. Hampsey, Microbiol. Mol. Biol. Rev. 62, 465 (1998); R. H. Reeder, Prog. Nucleic Acid Res. Mol. Biol. 62, 293 (1999); I. M. Willis, Eur. J. Biochem. 212, 1 (1993).
61. M. Hampsey, Microbiol. Mol. Biol. Rev. 62, 465 (1998); R. H. Reeder, Prog. Nucleic Acid Res. Mol. Biol. 62, 293 (1999); I. M. Willis, Eur. J. Biochem. 212, 1 (1993).
62. M. Hampsey, Microbiol. Mol. Biol. Rev. 62, 465 (1998); R. H. Reeder, Prog. Nucleic Acid Res. Mol. Biol. 62, 293 (1999); I. M. Willis, Eur. J. Biochem. 212, 1 (1993).
63. T. I. Lee and R. A. Young, Genes Dev. 12, 1398 (1998); M. Hampsey and D. Reinberg, Curr. Opin. Genet. Dev. 9, 132 (1999).
64. T. I. Lee and R. A. Young, Genes Dev. 12, 1398 (1998); M. Hampsey and D. Reinberg, Curr. Opin. Genet. Dev. 9, 132 (1999).
65. M. D. Rabenstein, S. Zhou, J. T. Lis, R. Tjian, Proc. Natl. Acad. Sci. U.S.A. 96, 4791 (1999).
66. D. Duboule, Ed., Guidebook to the Homeobox Genes (Oxford Univ. Press, New York, 1994).
67. I. G. Wool, Trends Biochem. Sci. 21, 164 (1996).
68. A. Lambertsson, Adv. Genet. 38, 69 (1998).
69. M. Jankowska-Anyszka et al., J. Biol. Chem. 273, 10538 (1998).
70. M. R. Culbertson, Trends Genet. 15, 74 (1999).
71. C. Burge, T. Tuschl, P. Sharp, in The RNA World, R. Gesteland, T. Cech, J. Atkins, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 2, 1999).
72. C. L. Will, C. Schneider, R. Reed, R. Luhrmann, Science 284, 2003 (1999).
73. R. Feyereisen, Annu. Rev. Entomol. 44, 507 (1999).
74. See D. Nelson's Web site (http://drnelson.utmem. edu/CytochromeP450.html).
75. G. von Heijne, J. Mol. Biol. 225, 487 (1992).
76. K. Hartenstein et al., Genetics 147, 1755 (1997).
77. R. G. Tearle, J. M. Belote, M. McKeown, B. S. Baker, A. J. Howells, Genetics 122, 595 (1989).
78. R. Malesika, Microbiology 143, 1781 (1997).
79. Q. Wang, G. Hasan, C. W. Pikielny, J. Biol. Chem. 274, 10309 (1999).
80. B. C. Dunkov and T. Georgieva, DNA Cell Biol. 18, 937 (1999).
81. T. Yoshiga et al., Eur. J. Biochem. 260, 414 (1999).
82. M. L. Kennard et al., EMBO J. 14, 4178 (1995).
83. High molecular weight genomic DNA was prepared from nuclei isolated [C. D. Shaffer, J. M. Wuller, S. C. R. Elgin, Methods Cell Biol. 44, 185 (1994)] from 2.59 g of embryos of an isogenic y; cn bw sp strain [B. J. Brizuela et al., Genetics 137, 803 (1994)]. The genomic DNA was randomly sheared, end-polished with Bal31 nuclease/T4 DNA polymerase, and carefully size-selected on 1% low- melting-point agarose. After ligation to BstX1 adaptors, genomic fragments were inserted into BstX1-linearized plasmid vector. Libraries of 1.8 ± 0.2 kb were cloned in a high-copy pUC18 derivative, and libraries of 9.8 ± 1.0, 10.5 ± 1.0, and 11.5 ± 1.0 kbp were cloned in a medium-copy pBR322 derivative. High-throughput methods in 384-well format were implemented for plasmid growth, alkaline lysis plasmid purification, and ABI Big Dye Terminator DNA sequencing reactions. Sequence reads from the genomic libraries were generated over a 4-month period using 300 DNA analyzers (ABI Prism 3700). These reads represent more than 12× coverage of the 120-Mbp euchromatic portion of the Drosophila genome (Table 1). Base-calling was performed using 3700 Data Collection (PE Biosystems) and sequence data were transferred to a Unix computer environment for further processing. Error probabilities were assigned to each base with TraceTuner software developed at Paracel Inc. (www.paracel.com). The predicted error probability was used to trim each sequence read such that the overall accuracy of each trimmed read was predicted to be >98.5% and no single 50-bp region was less than 97% accurate. The efficacy of TraceTuner and the trimming algorithm was demonstrated by comparing trimmed sequence reads to high-quality finished sequence data from BDGP (Fig. 2).
84. High molecular weight genomic DNA was prepared from nuclei isolated [C. D. Shaffer, J. M. Wuller, S. C. R. Elgin, Methods Cell Biol. 44, 185 (1994)] from 2.59 g of embryos of an isogenic y; cn bw sp strain [B. J. Brizuela et al., Genetics 137, 803 (1994)]. The genomic DNA was randomly sheared, end-polished with Bal31 nuclease/T4 DNA polymerase, and carefully size-selected on 1% low-melting-point agarose. After ligation to BstX1 adaptors, genomic fragments were inserted into BstX1-linearized plasmid vector. Libraries of 1.8 ± 0.2 kb were cloned in a high-copy pUC18 derivative, and libraries of 9.8 ± 1.0, 10.5 ± 1.0, and 11.5 ± 1.0 kbp were cloned in a medium-copy pBR322 derivative. High-throughput methods in 384-well format were implemented for plasmid growth, alkaline lysis plasmid purification, and ABI Big Dye Terminator DNA sequencing reactions. Sequence reads from the genomic libraries were generated over a 4-month period using 300 DNA analyzers (ABI Prism 3700). These reads represent more than 12× coverage of the 120-Mbp euchromatic portion of the Drosophila genome (Table 1). Base-calling was performed using 3700 Data Collection (PE Biosystems) and sequence data were transferred to a Unix computer environment for further processing. Error probabilities were assigned to each base with TraceTuner software developed at Paracel Inc. (www.paracel.com). The predicted error probability was used to trim each sequence read such that the overall accuracy of each trimmed read was predicted to be >98.5% and no single 50-bp region was less than 97% accurate. The efficacy of TraceTuner and the trimming algorithm was demonstrated by comparing trimmed sequence reads to high-quality finished sequence data from BDGP (Fig. 2).
85. For clone-based genomic sequencing, BAC, P1, and cosmid DNAs were prepared by alkaline lysis procedures and purified by CsCl gradient ultracentrifugation. DNA was randomly sheared and size-selected on LMP agarose for fragments in the 3-kb range for plasmids and in the 2-kb range for M13 clones. After blunt-ending with T4 DNA polymerase, plasmids were generated by ligation to BstX1 adaptors and insertion into BstX1-linearized pOT2A vector. M13 clones were generated using the double-adaptor protocol [B. Andersson et al., Anal. Biochem. 236, 107 (1996)]. Plasmid sequencing templates were prepared by alkaline lysis (Qiagen) or by PCR, and M13 templates were prepared using the sodium perchlorate-glass fiber filter technique [B. Andersson et al., Biotechniques 20, 1022 (1996)]. Paired end-sequences of 3-kb plasmid subclones were generated (principally) with ABI Big Dye Terminator chemistry on ABI 377 slab gel or ABI 3700 capillary sequencers. Additional M13 subclone sequence was generated using BODIPY dye-labeled primers. Procedures for finishing sequence to high quality at LBNL were as described (3).
86. For clone-based genomic sequencing, BAC, P1, and cosmid DNAs were prepared by alkaline lysis procedures and purified by CsCl gradient ultracentrifugation. DNA was randomly sheared and size- selected on LMP agarose for fragments in the 3-kb range for plasmids and in the 2-kb range for M13 clones. After blunt-ending with T4 DNA polymerase, plasmids were generated by ligation to BstX1 adaptors and insertion into BstX1-linearized pOT2A vector. M13 clones were generated using the double-adaptor protocol [B. Andersson et al., Anal. Biochem. 236, 107 (1996)]. Plasmid sequencing templates were prepared by alkaline lysis (Qiagen) or by PCR, and M13 templates were prepared using the sodium perchlorate-glass fiber filter technique [B. Andersson et al., Biotechniques 20, 1022 (1996)]. Paired end-sequences of 3-kb plasmid subclones were generated (principally) with ABI Big Dye Terminator chemistry on ABI 377 slab gel or ABI 3700 capillary sequencers. Additional M13 subclone sequence was generated using BODIPY dye-labeled primers. Procedures for finishing sequence to high quality at LBNL were as described (3).
87. M.-T. Yamamoto et al., Genetics 125, 821 (1990).
88. J. F. Abril and R. Guigo, Bioinformatics, in press.
89. A. Peter et al., in preparation.
90. J. Locke, L. Podemski, N. Aippersbach, H. Kemp, R. Hodgetts, in preparation.
91. The many participants from academic institutions are grateful for their various sources of support. We thank B. Thompson and his staff for the excellent laboratories and work environment, M. Peterson and his team for computational support, and V. Di Francesco, S. Levy, K. Chaturvedi, D. Rusch, C. Yan, and V. Bonazzi for technical discussions and thoughtful advice. We are indebted to R. Guigo and to E. Lerner of Aquent Partners for assistance with illustrations. The work described was funded by Celera Genomics, the Howard Hughes Medical Institute, and NIH grant P50-HG00750 (G.M.R.).