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A PAC/BAC End Sequencing Data Resource for Sequencing the Human Genome

Grant No.: DE-FC03-96ER62294
Principal Investigator: Pieter J. de Jong

Progress Report: 9/15/96- 7/7/97

The pilot project "A PAC/BAC End Sequencing Data Resource for Sequencing the Human Genome" has as it immediate goal the elucidation of the DNA sequence from the ends of PAC/BAC clones that comprise the current human genomic DNA libraries. Over the past ten months, our laboratory has set out to develop a high throughput DNA sequencing system based on the use of PCR-amplified clone ends as sequencing template. The initial four months of the project was directed toward the acquisition of the necessary capital equipment (two ABI 377 DNA Sequencers, MJ Research DNA Tetrad Thermal Cycler, Hamilton Microlab 2200 pipetting robot) and the hiring of technical personnel to perform the work. By incorporating a DOP-Vector PCR approach (Wu C.,et. al., 1996, NAR, 24:2614-2615.) to generate multiple copies of the clone ends, our laboratory has "rescued" approximately 7000 PAC clone ends during the past five months. These clone ends have been generated both randomly from the RPCI6 Human PAC library (about 4600 ends) and also a specific set of clone ends (approx. 2400 ends) covering a 3Mb region of chromosome 14 were sequenced from the RPCI1, 3, 4, 5 and 6 Human PAC Libraries. We have performed automated DNA sequencing using ET-dye labeled primers (Amersham) on these ends and have analyzed the data in detail. 2100 (30.1%) of the clone ends sequenced resulted in data that did not meet our minimum acceptable requirements of no more that 3 non-base calls in a 25 base window and/or less that 10% non-base calls in the remaining sequence. In addition, all "good" sequences must pass through Phred analysis and report greater than 20 to be considered as acceptable. The acceptable clone DNA end sequences (4900 ends) have an average Phred trimmed read length of 384 bases and an average Phred non-base call percentage of 4.25. More recent work on a much smaller set of clone ends (128) incorporating new dRhodamine dye terminator chemistry (ABI) indicates potential improvement in the quality of the data generated. For this set of clone ends the failure rate drops to 21.9%, the average read length increases to 443 bases while the percentage of non-base calls drops to 2.52%. Further work is in progress using the dRhodamine dye terminators to determine whether these results remain higher once a statistically significant number of clone ends has been sequenced. DNA sequence homology analysis (BLAST) indicates that 63.7% of the ends have unique sequence (no acceptable homology to known sequences in current data banks). 23.8% of the end sequences generated have homology to known EST's and STS's, while 3.2% identify with Line repeat elements and 9.3% to ALU repeat sequences. Detailed costs analysis has been performed based on our DNA sequencing success rate relative to the amount of research funds spent to date. When total dollars spent is compared to the total number of successful DNA end sequences generated, the costs per clone end is $72.79. After subtracting out all major equipment purchases and leases that were required to get the project off the ground, the cost drops to $42.45 per end. When only reagent and supply expenditures are considered, each end costs $22.66 to generate. The above costs analysis reflects the four month period when the DNA sequencing laboratory was being set up and essentially no data was being produced. In addition, two more months passed in which we were ramping-up our small scale techniques into high throughput PCR and DNA sequencing procedures. At our current level of production, we are able to generate successful DNA sequence from PCR end- rescued clone ends at a cost of $10.01 per end. This costs reflects all personnel, reagent and supply expenditures incurred but does not include any amortization of capital equipment employed in the project. At our current average read length this cost per clone end translates to a cost of $0.0261 per base.


Last modified: Wednesday, October 22, 2003

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