TRANSCRIPTOME 2002: From Functional Genomics to Systems
Bioinformatics for High-Throughput Mu Transposon Sequencing
Ursula Skalska, Yaron Butterfield, Ran Guin, Martin Krzywinski, Kim MacDonald, Duane Smailus, Jeff Stott, George Yang, Scott Zuyderduyn, Jacqueline Schein, Steven Jones and Marco Marra, Genome Sequence Centre BC Cancer Agency, Vancouver, BC, CANADA
As participants in the Mammalian Gene Collection full-length cDNA sequencing initiative, we have developed an efficient, high-throughput method for accurate sequencing of entire cDNA clone inserts. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Our approach uses a clone pooling strategy that eliminates the need for a transposon insertion library to be constructed for each clone. This process includes a number of key bioinformatic stages. Prior to pooling, analysis of Expressed Sequence Tags confirms clone identity. Accurate clone insert size and DNA quantitation data are used to ensure proportional representation of each cDNA clone in the pool. Sequences are assembled using Phred, Phrap, and Consed. Clones remaining incomplete are either re-pooled or finished using directed primer reads. Finished sequences are inspected for frameshifts and chimeric clones using BLAST and BLAT searches. Additionally, we have developed a number of programs to complement these technologies and to expedite sequencing of clones. Using these techniques, we have to date generated over 7.3 Mbp of accurate sequence from 3,891 cDNA clones. A detailed description of our strategy and the associated bioinformatics tools will be presented.
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