Systematic sequencing of cDNA clones using the transposon Tn5.
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.
Shevchenko
Yuriy
Y
NIH Intramural Sequencing Center, National Institutes of Health, Gaithersburg, MD 20877, USA.
Bouffard
Gerard G
GG
Butterfield
Yaron S N
YS
Blakesley
Robert W
RW
Hartley
James L
JL
Young
Alice C
AC
Marra
Marco A
MA
Jones
Steven J M
SJ
Touchman
Jeffrey W
JW
Green
Eric D
ED
D001482:Base Composition; D016010:Binomial Distribution; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D020161:Physical Chromosome Mapping / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004251:DNA Transposable Elements; D018076:DNA, Complementary