Chain Terminator Sequencing of Double-stranded DNA with Built-in Error Correction

Chain terminator sequencing is usually performed on single-stranded DNA. We propose a technique to determine a DNA sequence by sequencing both strands of a double strand in two four-lane gels. The first set of four lanes will contain a superposition of sequence information from the two complementary strands. The second set of four lanes will contain a different superposition in which one of the two single strands is "shifted" relative to the other. This technique provides redundant information sufficient for error correction—not just the error detection achieved by the standard approach of separately sequencing a single strand and its complement. The information content per four-lane gel is more than 50% higher in this double-stranded approach. Using this technique, the number of chain sequencings—including chain syntheses and gel separations—required to achieve a desired level of accuracy can be reduced by up to a factor of two. Unlike the ordinary single-stranded sequencing, some analysis is required to obtain the single-stranded sequence information from the double-stranded data, particularly in the presence of sequencing errors. This analysis and a computer simulation of double-stranded sequencing in the presence of errors are presented.