Sequence-tagged connectors: a sequence approach to mapping and scanning the human genome

Abstract

The sequence-tagged connector (STC) strategy proposes to generate sequence tags densely scattered (every 3.3 kilobases) across the human genome by arraying 450,000 bacterial artificial chromosomes (BACs) with randomly cleaved inserts, sequencing both ends of each, and preparing a restriction enzyme fingerprint of each. The STC resource, containing end sequences, fingerprints, and arrayed BACs, creates a map where the interrelationships of the individual BAC clones are resolved through their STCs as overlapping BAC clones are sequenced. Once a seed or initiation BAC clone is sequenced, the minimum overlapping 5' and 3' BAC clones can be identified computationally and sequenced. By reiterating this "sequence-then-map by computer analysis against the STC database" strategy, a minimum tiling path of clones can be sequenced at a rate that is primarily limited by the sequencing throughput of individual genome centers. As of February 1999, we had deposited, together with The Institute for Genomic Research (TIGR), into GenBank 314,000 STCs ( approximately 135 megabases), or 4.5% of human genomic DNA. This genome survey reveals numerous genes, genome-wide repeats, simple sequence repeats (potential genetic markers), and CpG islands (potential gene initiation sites). It also illustrates the power of the STC strategy for creating minimum tiling paths of BAC clones for large-scale genomic sequencing. Because the STC resource permits the easy integration of genetic, physical, gene, and sequence maps for chromosomes, it will be a powerful tool for the initial analysis of the human genome and other complex genomes.

Figure 1

A model for the virtual genome map generated by the STC resource. The sequence of a nucleation (seed) BAC clone reveals overlapping BAC clones through a search of the STC database with this sequence. The minimal overlapping 5′ and 3′ BAC clones can then be chosen for sequence extension. As this process is reiterated, the virtual genome map becomes explicit.

Figure 2

STC map across the 1.1-Mb human α/δ T cell receptor locus. This diagram depicts a BAC contig constructed by using a 1,071-kb sequence from the T cell receptor α locus to query the STC database. The sequence was masked for repeats (http://repeatmasker.genome.washington.edu/) and used to search the TIGR STC database (http://www.tigr.org/tdb/humgen/bac_end_search/bac_end_search.html) which, at the time, contained around 310,000 sequences. BAC clones shown as solid lines had both end sequences available in the STC database; dotted lines represent BAC clones where only one end sequence was available The arrow-head points in the direction of the unsequenced end, and the insert size is arbitrarily set at 150 kb. The bottom scale depicts the length in kb of this locus.