What is the strategy for full-insert sequencing?
It is a directed approach, based on a commercially available in vitro transposition system. The insert size of all 5,849 DGC clones have been determined and sorted into four size categories (<1.4 kb, 1.4-3.0 kb, 3.0-4.5 kb, and >4.5 kb). We estimate that 72% of the DGC clones(1.4-4.5 kb) will be sequenced from mapped PCR products, 23%(<1.4 kb) will be sequenced by oligo walking, and 4%(>4.5 kb) will be sequenced from plasmid prepared template derived from non-mapped transposon bearing cDNAs. Experiments suggest 70-80% of those clones that receive transposons will finish to high quality without any extra work. The remaining 20-30% will be finished to high quality using custom oligos. This in vitro system has a number of advantages that we prefer to other commercial in vitro systems. One major feature is that it allows us to sequence directly from the mapped transposon PCR products using nested primers. Using PCR products as sequencing templates provides a significant cost reduction in c
It is a directed approach, based on a commercially available in vitro transposition system. The insert size of all 5,849 DGC clones have been determined and sorted into four size categories (<1.4 kb, 1.4-3.0 kb, 3.0-4.5 kb, and >4.5 kb). We estimate that 72% of the DGC clones(1.4-4.5 kb) will be sequenced from mapped PCR products, 23%(<1.4 kb) will be sequenced by oligo walking, and 4%(>4.5 kb) will be sequenced from plasmid prepared template derived from non-mapped transposon bearing cDNAs. Experiments suggest 70-80% of those clones that receive transposons will finish to high quality without any extra work. The remaining 20-30% will be finished to high quality using custom oligos. This in vitro system has a number of advantages that we prefer to other commercial in vitro systems. One major feature is that it allows us to sequence directly from the mapped transposon PCR products using nested primers. Using PCR products as sequencing templates provides a significant cost reduction in c
