Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project Mark D, Adams; Jenny M. Kelley; Jeannine D. Gocayne; Mark Dubnick; Mihael H. Polymeropoulos; Hong Xiao; Carl R. Merril; Andrew Wu; Bjorn Olde; Ruben F. Moreno; Anthony R. Kerlavage; W. Richard MeCombie; J. Craig Venter Science, New Series, Vol. 252, No, 5013 (Jun. 21, 1991), 1651-1656. Stable URL: hhup/finks jstor.orgh 'sici=0036-8075%2819910621%293%3A252%3A5013403C 165 1%3ACDSEST%3E2.0,CO%3B2-6 Science is currently published by American Association for the Advancement of Science, ‘Your use of the ISTOR archive indicates your acceptance of JSTOR’s Terms and Conditions of Use, available at hhup:/www.jstororg/about/terms.hml. JSTOR’s Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at hup:/www jstor-org/journals/aaas.html, Each copy of any printed page of su wt of a JSTOR transmission must contain the same copyright notice that appears on the sereen or transmission, JSTOR is an independent not-for-profit organization dedicated to ercating and preserving a digital archive of scholarly journals. For more information regarding JSTOR, please contact support @ jstor.org. hupulwww jstor.org/ Tue Nov 8 15:10:30 200887, ER, Fewon oa, Sco 247, 49 (1990), AR. KW. Rin al shi 281, 1866 (1991), 89, §Srnavn, 2 Zo K.P, W. Bate, E. H. Chang, Nate 348,747 (0990) 90, 8. Kwok an J.J. Sina, PR Tel: Pres and Appts fr DNA Anplftaion Ti Bech, 8. (Sttom, New York, 1989), pp 255-244, 91. Tf White, Mad). H.Pering, Ci: Chon rea, D. Wiliams tnd 8. Kwok, in Lavatory Digi of Vin! Ife, He Lemme, El (Debher, New Yk, 191) p. 187-173 PS. Wik, D- A Masger Be Higck, Bote 10, 56 (191), Mike, J Mibalovet Rc iguch .S Wabi, Enh unpulced data J Bllera J sp. he.168, 115 (1989); B. Boole el AJ. Ham. ee 48, 157 99), PRS 98. RC Alln,G. Graves, B. Budo, Bivens 7,736 (1989); K. Kase, J. Foes 8 35, 196 (1950). 96.8. Scart unpubl da 97. R elo ea Am J Ham. Govt 47,518 (199). 98. M, Stocking, D Hedgecock, R. Higych Vigan, HA. ech ii. 4370 (1) 99, We thnk oe collegues who conte the development and apn of PGR. The space conuoin of thie rose and the many pubes on PCR Prevents comprehensive rey of ances a pple we pg any {farses wae fn x br ge, Weare pa IRSukt 8 Schr. Higch, and Abramon for allowing te tr Spubloied work Rexel review; and K- Leena pepsin of oem Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project Mark D. ApaMs, JENNY M. KELLEY, JEANNINE D. GocaYNE, Mark ‘DUBNICK, Mirvac H. PoryMERoPouLos, HONG XIAO, CARL R. MERRIL, ANDREW WU, ByoRN OLDE, RUBEN F. MorENO, ANTHONY R. KERLAVAGE, W. RicHarp McComptg, J. CRAIG VENTER* Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain comple- mentary DNA (cDNA) clones to generate expressed se- quence tags (ESTs). ESTs have applications in the discov- «ery of new human genes, mapping of the human genome, and “Hentfeation of coding regions in_ genomic se ces. OF the sequences generated, 337 new a a at eae cee sm other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of spit; and a murine tyrosine kinase . Forty-six ESTS ee ea polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields. t0 100,000 genes, up to 30,000 of which may be expressed, in the brain (1). However, GenBank lists the sequence of only a few thousand human genes and <200 human brain messen- ger RNAs (mRNAs) (2). Once dedicated human chromosome T HE HUMAN GENOME IS ESTIMATED TO CONSIST OF 50,000 MD. duns, JM. Kelley, J.D. Gray, M, Dab, A.W, B, Oe, Moreno, AR: Kesnage W. Ie MeComb, and JC. Vener re the Sco ‘Resp’ Binchemy and Molccuar log, Nason Inne of Newologa Daordes and Sue, Nason Lotus of Heh, Bethesda, MD 20892, NH ogmergn HX aad CR Mera in he Ito Bashi Gener Nol Tne of Mena Heath, Neurscenee Cero Se Elzabes Hosp, Washington DO 50032 “Fo whom corerpondenc shoud be addres a 2170NE 1991 sequencing begins in § yeats, itis expected that 12 to 15 years will bbe required to complete the sequence of the genome (3). It is therefore likely that the majority of human genes will remain tunknown for at least the next decade. The merits of sequencing DNA, reverse transcribed from mRNA, as a part of the human, {genome project have been vigorously debated since the idea of determining the complete nucleotide sequence of humans first surfaced. Proponents of cDNA sequencing have argued that because the coding sequences of genes represent the vast majority of the information content of the genome, but only 3% of the DNA, ‘eDNA sequencing should take precedence over genomic sequencing (4). Proponents of genomic sequencing have argued the difficulty of finding every mRNA expressed in al issues, cll types, and devel- ‘opmental stages and have pointed out that much valuable informa- tion from intronic and intergenic regions, including control and regulatory sequences, will be missed by cDNA sequencing (3). However, many genome enthusiasts have incorrectly stated that ‘gene coding regions, and therefore mRNA sequences, ae readily predictable from genomic sequences and have concluded that there is no need for large-scale CDNA sequencing. In fact, prediction of ‘transcribed regions of human genomic sequence is currently feasible ‘only for relatively large exons (6). ‘On the basis of our high output with automated DNA sequence analysis of 96 templates per day and consideration of the above issues, we initiated a pilot project to test the use of partial CDNA. sequences (ESTS) in a comprehensive survey of expressed genes. Sequence-tagged sites (STSs) are becoming standard markers for ‘the physical mapping of the human genome (7). These short sequences from physically mapped clones represent uniquely iden tified map positions, ESTs can serve the same purpose a the random ‘genomic DNA STS¢ and provide the additional feature of pointing directly to an expressed gene. An EST is simply a segment of a sequence from a cDNA clone that corresponds to an mRNA. ESTs longer than 150 bp were found to be the most useful for similarity searches and mapping. ARTICLES 1661Libraries of Complementary DNAs (Of the estimated 30,000 genes expressed in the human brain, as ‘many a5 20,000 may encode low-abundance, brain-pecific tran- scripts (1), The fat that up to one-fourth of al genetic diseases affect neurological functions is an indication of the diversity and impor- tance of genes expressed in the brain (8) ‘An assumption in our choice of cDNA libraries was that random primed and partial cDNA clones would be more informative in ‘identifying genes and constructing a useful EST database than sequencing from the ends of full-length cDNAs (which contain 5° and 3° untranslated sequences) would be. By obtaining coding Sequences, we hoped t0 take advantage of more sensitive peptide comparisons, in addition to nucleotide sequence comparisons. To discover the inherent limitations to be overcome in a large-sale cDNA sequencing project, we wanted to examine the diversity of representative cDNA libraries, identify desirable and undesirable characteristics of the libraries, and determine the information content and accuracy of single-run sequencing from both coding, and flanking regions. Single-run sequencing involves performing a single sequence reaction, rather than relying on multiple, red: dant reactions from each strand. We chose three commercial human brain cDNA libraries made from mRNA isolated from the hippocampus and temporal cortex ofa 2-year-old female and from a feral brain (9). ‘Single-ran DNA sequence data were obtained from 609 random- ly chosen cDNA clones (Table 1). Double-stranded cDNA clones in the pBluescript vector (Stratagene) were sequenced by a cycle sequencing protocol (10) with dye-labeled primers and 373A DNA Sequencers (Applied Biosystems). The average length of usable sequence was 397 bases with a standard deviation of 99 bases. ‘Subtracive hybridization has been used by researchers to reduce the population of highly represented sequences in a ¢DNA library (11, 12) by selectively removing sequences shared by another library We tested. subtractive hybridization as a way of enhancing the ‘number of brain-specific clones in the hippocampus library by hybridizing the hippocampus library with a WI38 human hung, fibroblast cell line cDNA library and removing the common se quences (Table 1) (12, 13). Table 1. Competition of feo) das) a) pa ma Ser 2 ito) 7 {eo $ fisy Bo) No dunn mach 1 cas) 4 9) 20 (29) 8 03) Bijele ee ss Gan) 2 (07 0 “Gy GH Nome 1 a3} 3 83 ao % (3 vst scuevee, vou 282