DNA sequencing by hybridization using semi-degenerate bases.

One way to enhance the performance of hybridization microarrrays for DNA de novo sequencing is the use of probing patterns with gaps of unsampled positions. Ideally, such gaps could be realized by the inclusion into microarray oligos (probes) of wild-card compounds, referred to as universal bases (which bind nonspecifically to natural bases). The suggested alternative is to deploy in the gap positions degenerate bases, i.e., uniform mixtures of the four natural bases, with ensuing deterioration of the hybridization signal. In this paper, we show that such signal loss is a minor shortcoming, compared with the fact that degenerate bases cannot be treated as universal. Indeed, the substantial spread of hybridization energies at any microarray feature is such that on overwhelming number of mismatches bind more strongly than legal matches. We observed, however, that much narrower energy spreads are exhibited by pairs of bases in the same strength class (A-T and C-G). We call semi-degenerate a gap position realized with bases in the same energy class and show that well-known sequence reconstruction algorithms can be modified to achieve substantial improvements in sequencing effectiveness. For example, with a 4(9)-feature microarray and an acceptable weakening of the hybridization signal, one may achieve lengths of about 4,000 bases (compared with < 250 of the standard uniform method). Our approach also incorporates the use of a spectrum expressed in terms of observed feature melting temperatures (analog spectrum), rather than binary decisions made directly at the biochemical level (digital spectrum). While universal bases represent the ultimate goal of sequencing by hybridization, semidegenerate natural bases are the most effective known substitute.