Nucleic Acids Research, 2002, Vol. 30, No. 11 2469-2477
© 2002 Oxford University Press
Systematic sequencing of cDNA clones using the transposon Tn5
1NIH Intramural Sequencing Center, National Institutes of Health, Gaithersburg, MD 20877, USA, 2Genome Technology Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA, 3Genome Sciences Centre, BC Cancer Research Centre, Vancouver, BC V5Z 4E6, Canada and 4Invitrogen Corporation, Rockville, MD 20850, USA
In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale ‘shotgun-style’ sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.
* To whom correspondence should be addressed at: NIH, 50 South Drive, Building 50, Room 5523, Bethesda, MD 20892, USA. Tel: +1 301 402 0201; Fax: +1 301 402 4735; Email: egreen{at}nhgri.nih.gov Present address:James L. Hartley, Protein Expression Laboratory, National Cancer Institute, Frederick, MD 21702, USA
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Qiao, Y. Sun, J. Qiao, and L. Mindich Temporal Control of Message Stability in the Life Cycle of Double-Stranded RNA Bacteriophage {Phi}8 J. Virol., January 15, 2009; 83(2): 633 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Lyell, A. K. Dunn, J. L. Bose, S. L. Vescovi, and E. V. Stabb Effective Mutagenesis of Vibrio fischeri by Using Hyperactive Mini-Tn5 Derivatives Appl. Envir. Microbiol., November 15, 2008; 74(22): 7059 - 7063. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. TURNER, C. M. NORMAN, M. J. CHURCHER, and A. J. NEWMAN Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome RNA, March 1, 2006; 12(3): 375 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-L. BOON, C. M. NORMAN, R. J. GRAINGER, A. J. NEWMAN, and J. D. BEGGS Prp8p dissection reveals domain structure and protein interaction sites RNA, February 1, 2006; 12(2): 198 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kawano, T. Yoshida, K. Hieda, J. Aoki, H. Miyoshi, and Y. Koyanagi A Lentiviral cDNA Library Employing Lambda Recombination Used To Clone an Inhibitor of Human Immunodeficiency Virus Type 1-Induced Cell Death J. Virol., October 15, 2004; 78(20): 11352 - 11359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Baross, Y. S.N. Butterfield, S. M. Coughlin, T. Zeng, M. Griffith, O. L. Griffith, A. S. Petrescu, D. E. Smailus, J. Khattra, H. L. McDonald, et al. Systematic Recovery and Analysis of Full-ORF Human cDNA Clones Genome Res., October 1, 2004; 14(10b): 2083 - 2092. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R. Herron, G. Hughes, G. Chandra, S. Fielding, and P. J. Dyson Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA Nucleic Acids Res., August 12, 2004; 32(14): e113 - e113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kang, T. Durfee, J. D. Glasner, Y. Qiu, D. Frisch, K. M. Winterberg, and F. R. Blattner Systematic Mutagenesis of the Escherichia coli Genome J. Bacteriol., August 1, 2004; 186(15): 4921 - 4930. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. Kazazian Jr. Mobile Elements: Drivers of Genome Evolution Science, March 12, 2004; 303(5664): 1626 - 1632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Y. Goryshin, T. A. Naumann, J. Apodaca, and W. S. Reznikoff Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis Genome Res., April 1, 2003; 13(4): 644 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Mammalian Gene Collection Program Team*, R. L. Strausberg, E. A. Feingold, L. H. Grouse, J. G. Derge, R. D. Klausner, F. S. Collins, L. Wagner, C. M. Shenmen, G. D. Schuler, et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences PNAS, December 24, 2002; 99(26): 16899 - 16903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Naumann, I. Y. Goryshin, and W. S. Reznikoff Production of combinatorial libraries of fused genes by sequential transposition reactions Nucleic Acids Res., November 1, 2002; 30(21): e119 - e119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. S. N. Butterfield, M. A. Marra, J. K. Asano, S. Y. Chan, R. Guin, M. I. Krzywinski, S. S. Lee, K. W. K. MacDonald, C. A. Mathewson, T. E. Olson, et al. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones Nucleic Acids Res., June 1, 2002; 30(11): 2460 - 2468. [Abstract] [Full Text] [PDF]
|
|