Nucleic Acids Research, 2002, Vol. 30, No. 11 2460-2468
© 2002 Oxford University Press
An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones
Genome Sciences Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada
We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.
* To whom correspondence should be addressed. Tel: +1 604 877 6084; Fax: +1 604 877 6085; Email: mmarra{at}bcgsc.bc.ca
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. D. Morin, E. Chang, A. Petrescu, N. Liao, M. Griffith, R. Kirkpatrick, Y. S. Butterfield, A. C. Young, J. Stott, S. Barber, et al. Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling Genome Res., June 1, 2006; 16(6): 796 - 803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Yu and D. V. Schaffer Selection of novel vesicular stomatitis virus glycoprotein variants from a Peptide insertion library for enhanced purification of retroviral and lentiviral vectors. J. Virol., April 1, 2006; 80(7): 3285 - 3292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Smailus, A. Marziali, P. Dextras, M. A. Marra, and R. A. Holt Simple, robust methods for high-throughput nanoliter-scale DNA sequencing Genome Res., October 1, 2005; 15(10): 1447 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Pajunen, A. T. Pulliainen, J. Finne, and H. Savilahti Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes Microbiology, April 1, 2005; 151(4): 1209 - 1218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zhang, D. Kitsberg, H. Chy, Q. Zhou, and J. R. Morrison Transposon-mediated generation of targeting vectors for the production of gene knockouts Nucleic Acids Res., February 7, 2005; 33(3): e24 - e24. [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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Vilen, J.-M. Aalto, A. Kassinen, L. Paulin, and H. Savilahti A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes J. Virol., December 6, 2002; 77(1): 123 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shevchenko, G. G. Bouffard, Y. S. N. Butterfield, R. W. Blakesley, J. L. Hartley, A. C. Young, M. A. Marra, S. J. M. Jones, J. W. Touchman, and E. D. Green Systematic sequencing of cDNA clones using the transposon Tn5 Nucleic Acids Res., June 1, 2002; 30(11): 2469 - 2477. [Abstract] [Full Text] [PDF]
|
|