A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides.

摘要

Direct sequencing of cosmid DNAs using a library of oligonucleotide primers of length 8, 9, or 10 is proposed. The statistics of priming indicate that a primer library sufficient for determining the sequence of the entire human genome (100,000 cosmids) would be small enough to be assembled and managed. Such a library would greatly reduce the cost and effort of high-volume sequencing: primers would be instantly available; the sequence of each cosmid DNA could be determined from a single DNA preparation without the necessity for mapping or subcloning; and, because each primer would be used repeatedly, the cost of primers would become a negligible fraction of other costs. A combination of random and directed priming could determine the sequence of a cosmid DNA in 1.2-1.5 times the minimum number of sequencing reactions required, and completely directed priming would be even more efficient. The success of this strategy requires that a considerable fraction of octamers, nonamers, or decamers be able to prime selectively in double-stranded DNAs 45,000 base pairs (bp) long; initial results indicate that this is likely to be the case. The strategy is not limited to cloned DNAs and would be useful for rapid identification and direct sequencing of viral nucleic acids.