Peak Height Pattern in Dichloro-Rhodamine and Energy Transfer Dye Terminator Sequencing

Abstract

Establishing the pattern in peak heights within local sequence contexts improves the accuracy of base calling and the identification of DNA sequence variations in dye-terminator cycle sequencing. We have systematically examined pairs of sequence-tagged sites (STSs) that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by two recently commercialized dye-terminator chemistries, the dichloro-rhodamine (dRhodamine) and the energy transfer (BigDye^TM) terminators. For sequencing traces generated with the dRhodamine terminators, the peak height of a particular base in 28 of 64 possible 3-base windows (44%) can be predicted by knowing just one or two bases 5′ to the base in question. For those generated with the BigDye terminators, the peak height of a particular base in 23 of 64 possible 3-base windows (36%) can be predicted by knowing the local sequence context. When the peak heights are binned slightly differently, 75% (48 out of 64 cases) of the base peaks generated by both dRhodamine and BigDye terminators fall in the middle half, confirming that the peak patterns of these two new dye terminator chemistries are much more even than those found in the original rhodamine dye terminator sequences.