Glenda G. Quan, Karolyn Burkhart-Shultz, Timothy Andriese, Andre Arellano, Long Do, Arthur Kobayashi, Brent Kronmiller, Madison Macht, Matt Nolan, David Ow, Hoan Phan, Melissa Ramirez, Warren Regala, Christina Sanders, Stephanie Stilwagen, Astrid Terry, Nelson Velasco, Vijay Viswanathan, Anthony V. Carrano, and Jane E. Lamerdin 
Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550 
quan1@llnl.gov 

The goal of finish sequencing is to obtain high-quality, contiguous sequence of cosmid and BAC clone inserts. A major component of finish sequencing is gap closure. In order for the sequence to be contiguous, gaps in the initial sequence data, obtained from random shotgun sequencing, must be closed. At the Joint Genome Center at Lawrence Livermore National Laboratory, we employ three main strategies for sequence gap closure: transposon "bombing", shatter library production, and custom primer walking. We currently use an in vitro transposon insertion strategy involving the random insertion of a yeast transposable element into a gap-spanning, circular plasmid. Using primers designed off both ends of the transposable element, new sequence can be obtained directing away from the insertion point. Transposon "bombing" allows us to identify new sequencing start points within the gap itself, and gives us the advantage of sequencing with two primers. In the shatter method, a double-stranded, linear fragment containing the gap sequence (e.g. a PCR product or restriction fragment) is sonicated into fragments of 300-500 bp in length. These short fragments are then sub-cloned into an M13 phage vector and sequenced using conventional ET-forward primers. These shatter libraries are particularly well-suited to regions of significant secondary structure which are recalcitrant to conventional sequencing chemistries, where the smaller inserts may contain only a portion of the hairpin in the original gap-spanning clone. Additionally, the data generated by these clones are very high in quality and can be assembled as 'mini' shotgun projects in those instances of very difficult assembly problems, such as long tandem repeats. Our third strategy utilizes the automated primer picking program in the sequence editor Consed for primer walking on existing clones that span a gap. The main advantage of primer walking is that it allows closure of small gaps with a minimum number of sequencing reads. We have used various combinations of these three strategies to increase our output of finished sequence by over 500% in the last fiscal year. Analyses are underway to evaluate the efficiency and cost of these three strategies in order to better tailor automated finishing protocols needed to achieve the ambitious sequencing ramps required to complete the JGI's portion of the human genome. 

This work was performed by Lawrence Livermore National Laboratory under the auspices of the U.S. Department of Energy, Contract No. W-7405-Eng-48.