An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones.
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.
Butterfield
Yaron S N
YS
Genome Sciences Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada.
Marra
Marco A
MA
Asano
Jennifer K
JK
Chan
Susanna Y
SY
Guin
Ranabir
R
Krzywinski
Martin I
MI
Lee
Soo Sen
SS
MacDonald
Kim W K
KW
Mathewson
Carrie A
CA
Olson
Teika E
TE
Pandoh
Pawan K
PK
Prabhu
Anna-Liisa
AL
Schnerch
Angelique
A
Skalska
Ursula
U
Smailus
Duane E
DE
Stott
Jeff M
JM
Tsai
Miranda I
MI
Yang
George S
GS
Zuyderduyn
Scott D
SD
Schein
Jacqueline E
JE
Jones
Steven J M
SJ
D010583:Bacteriophage mu / Q000235:genetics*; D001482:Base Composition; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D009010:Monte Carlo Method; D016254:Mutagenesis, Insertional / Q000235:genetics*; D020161:Physical Chromosome Mapping / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013379:Substrate Specificity; D013997:Time Factors
D017931:DNA Primers; D004251:DNA Transposable Elements; D018076:DNA, Complementary