Sequence Ready—or Not?

En route to the goal of the Human Genome Project (HGP) of obtaining the complete sequence of the human genome by the year 2005, several milestones have been reached (Collins and Galas 1993). First was the construction of a genetic linkage map with an average resolution of 1 cM (Dib et al. 1996). Second was the construction of a physical map with an average marker spacing of <150 kb (Hudson et al. 1995). These are laudable achievements; however, they only represent the completion of the initial phase of generating a sequenceready physical map of the human genome that will provide the necessary templates for large-scale sequencing. The current physical map is composed of two main resources. The first is physical maps made up of overlapping cloned fragments of human DNA. These clone contigs have been largely constructed using sequence-tagged site (STS) content mapping methods (Green and Olson 1990). The most comprehensive human genome physical maps are largely composed of overlapping yeast artificial chromosome [(YAC) Burke et al. 1992] clones [see http: / /wwwgenome.wi.mit.edu (Cohen et al. 1993; Chumakov et al. 1995; Hudson et al. 1995)]. Unfortunately, YAC clones are not suitable substrates for large-scale sequencing of the human genome. This is largely due to the high rate of chimaerism, the high frequency of deletions and rearrangements observed in these clones, and the difficulty of obtaining purified YAC DNA. The second physical map resource that is widely used is the whole-genome radiation hybrid (RH) map (Hudson et al. 1995; Schuler et al. 1996; Stewart et al. 1997). This map provides many additional ordered and binned markers and has been integrated with the YAC map. It is important to realize that although the average STS spacing is approximately one every 150 kb, the distribution is not random. A large number of these markers have been derived for positional cloning projects or from expressed sequence tags (ESTs), which may enrich marker density in disease regions or gene-rich regions, respectively. The combined maps provide much of the basis for the current effort to generate a sequence-ready map of the human genome. A sequence-ready map must meet certain criteria. Obviously, it must be composed of clones that are suitable sequence templates. Experience with sequencing the yeast and Caenorhabditis elegans genomes has shown that bacterial clones are an excellent choice. The primary clones being used are bacterial artificial chromosomes [(BACs) Shizuya et al. 1992], P1-derived artificial chromosomes [(PACs) Ioannou et al. 1994], and cosmids (Wahl et al. 1987). The clones corresponding to the STSs from a target region are typically isolated from arrayed libraries using PCR or hybridization strategies (see Fig. 1). An alternate approach is to subclone YAC clones into cosmids directly (Wong et al. 1997). Once the appropriate clones have been identified, the next step is to assemble these clones into an ordered contig. Clones are subjected to restriction endonuclease digestion, and fragments are separated by gel electrophoresis (Marra et al. 1997; Wong et al. 1997). Overlapping clones are then identified by shared restriction pattern or ‘‘fingerprint’’ similarities (see Fig. 2). It is a critical requirement for a sequence-ready map that the contigs have sufficient depth so that all restriction fragments can be validated by several overlapping clones. That is, if restriction fragment inconsistencies are observed, enough clones must be examined to establish the correct fragment pattern from the anomalous one. This is further complicated by the presence of polymorphisms. The BAC and PAC libraries being used for assembling sequence-ready maps of the human genome have two, and often more, haplotypes. Experience has shown that 6to