Systematic sequencing of cDNA clones using the transposon Tn5

Nucleic Acids Res. 2002 Jun 1;30(11):2469-77. doi: 10.1093/nar/30.11.2469.


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.

MeSH terms

  • Base Composition
  • Binomial Distribution
  • Cloning, Molecular
  • DNA Transposable Elements / genetics*
  • DNA, Complementary / genetics*
  • Genetic Vectors / genetics
  • Mutagenesis, Insertional / genetics
  • Physical Chromosome Mapping / methods
  • Recombination, Genetic / genetics
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*


  • DNA Transposable Elements
  • DNA, Complementary