Christopher H. Martin, Cheryl A. Davis, Cheryl L. Ericsson, Carol A. Mayeda, Herb Moise and Michael J. Palazzolo
Human Genome Center, Lawrence Berkeley Laboratory, Berkeley, CA 94720.
Our group has developed a novel directed approach to genomic sequencing in which every sequencing template is mapped to a resolution of 30 base pairs prior to being sequenced. This high resolution mapping information yields two important advantages. First, genomic sequence can be determined with far fewer sequencing reactions than approaches which utilize random coverage to yield the bulk of the complete sequence. Second, the difficulty of the sequence assembly problem is greatly reduced, as this high resolution physical mapping information provides significant guidance to the process of reconstruction of the original genomic sequence.
Using our directed approach, we have completed over 1,000,000 base pairs of completely double stranded and edited human genomic sequence. Our initial sequencing target in the human genome consists of a large growth factor rich region located at 5q31-q35. All of this sequence is derived from sequencing subclones derived from P1 physical mapping clone sets that span this region. We have found the P1 physical mapping system to be an excellent substrate for our genomic sequencing efforts. This megabase of sequence is all of high quality (less than 1 error per 2,500 base pairs and all completely double stranded) and was completed during the past two years. Based on these achievements, we are now in the process of scaling up our efforts in human genomic DNA sequencing at LBNL.
In addition to sequence production efforts, a major focus of the center is on technology development, where our goal is the continuing improvement of the directed sequencing strategy. In the area of biology, we have recently replaced an agarose/PCR based step in our high resolution physical mapping phase with one based on sequencing of the ends of several ~3 kb subclones. In automation, a new integrated platform for PCR reaction setup and subsequent thermocycling is the entering of the production testing phase. In the area of informatics, we are beginning to use new tools being developed both at the LBNL Center and also in collaboration with Gene Meyers at the University of Arizona. These tools capture and reflect the high resolution physical mapping information made available by the directed sequencing strategy for sequence assembly and automation of many sequence finishing decisions. Overall, we are continuing to make progress towards our goal of using the directed strategy as a biological platform for the development of a highly automated system for the sequencing of genomic DNA.