Exploring DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data

Abstract

Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA structure. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.\n\nAuthor SummaryDNA can be found in various forms that differ from the double-helix first discovered by Watson and Crick in 1953. These alternative DNA structures depend on the DNA sequence, and researchers continue to explore which sequences have the potential to form alternative structures. Here we advance the use of Pacific Biosciences sequencer data to explore potential alternative DNA structures. The Pacific Bio-sciences sequencer provides an unprecedented way to examine the interaction between DNA polymerase and DNA by following a single polymerase in real time as it copies a DNA molecule. The pausing of DNA polymerase is a common method for exploring the DNA sequences that have the potential to form alternative DNA structures, and Pacific Biosciences data has previously been used to measure polymerase pausing at a slipped strand structure. DNA polymerase is known to pause at some of these alternative structures, such as the structure known as the G-quadruplex, a DNA structure that has potentially importing regulatory significance. We examine polymerase kinetics around a G-quadruplex, and find evidence of polymerase pausing in the Pacific Biosciences kinetics. We provide a method, with publicly available code, so that others can examine these polymerase kinetics for any sequence of interest.