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 (Ϸ135 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 largescale 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.

Contigs Built with Fingerprints, Markers, and FPC V4.7

“…The majority of BAC clones from the RPCI-11 library have been fingerprinted at the GSC in St. Louis, and these are supplemented with additional clones fingerprinted at the Sanger Centre by using the same protocol so that the fingerprints are compatible (Marra et al 1997;Humphray et al 2000). In order to extend the contigs by walking, STSs are designed using the available GSS (Genome Sequencing Survey; Mahairas et al 1999) from selected clones at the ends of contigs. These STSs then are used to identify joins between existing contigs or to identify additional BAC clones from the library.…”

End-sequence profiling: Sequence-based analysis of aberrant genomes

“…ESP takes advantage of efficient techniques for BAC library construction (14,15) and BAC end sequencing (6,16) developed in support of the human genome project, and it fully uses the nearly complete human genome sequence (17,18). Still, ESP might seem too expensive and labor intensive to be practical.…”

“…End-sequence profiling (ESP) as described here complements these techniques by providing high-resolution copy number and structural aberration maps on selected disease tissues. ESP is based on the concept of sequence-tagged connectors developed to facilitate de novo genome sequencing (6). We chose the MCF-7 breast cancer cell line as a demonstration system for ESP because the line was assessed by using both CGH and spectral karyotyping and is remarkable in its complexity (7).…”

A Personal View of Molecular Technology and How It Has Changed Biology

“…34,35 This approach was based on our earlier theoretical analyses of optimal strategies through paired-end insert sequencing for analyzing the human genome. [35][36][37] The idea is to sequence both ends of 150 000 randomly generated bacterial artificial chromosome (BAC) inserts (average 200 kilobases (kb) in length) from the human genome. This would give a 500 base pair (bp) tag about every 10 kb across the genome.…”

“…This is an enormously efficient approach to sequencing genomes. Greg Mahairas sequenced the first draft of the rice genome using the STC approach 35 as a collaboration with Monsanto.…”