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- Title
Systematic sequencing of cDNA clones using the transposon Tn5.
- Authors
Shevchenko, Yuriy; Bouffard, Gerard G; Butterfield, Yaron S N; Blakesley, Robert W; Hartley, James L; Young, Alice C; Marra, Marco A; Jones, Steven J M; Touchman, Jeffrey W; Green, Eric D
- Abstract
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.
- Publication
Nucleic acids research, 2002, Vol 30, Issue 11, p2469
- ISSN
1362-4962
- Publication type
Journal Article
- DOI
10.1093/nar/30.11.2469