Ordered DNA Fragmentation and Amplification for NGS Applications

In this study, the enzymatic in situ cutting of linearized DNA molecules at approximately 11kbp intervals is demonstrated using a soft lithography technique. The ultimate goal is to provide a general ordered cutting method to greatly simplify the assembly process of NGS systems by reducing computational reassembly complexity. DNA was stretched onto PMMA (Poly methyl methacrylate) coated silicon by withdrawing the substrate from a DNA solution (a process termed “combing”). The stretched lambda DNA could be linearly cut with a soft lithography stamp used to selectively apply DNase I. After cutting the DNA on the substrate, the DNA fragments are removed from the surface by incubating PMMA in the commercial NEBuffer 3.1 at 75°C. The recovered fragments desorbed into the buffer and were sequenced using the PacBio RS II sequencer without an amplification step. The mean coverage was 2870X for the approximately 11 kbp fragmented sample and 100% of the lambda genome was sequenced. Methods to extend of the technique to ordered fragmentation are discussed.