(346c) DNA Sample Preparation for Long-Read Genomics Using Microfluidics

Genome analysis is rapidly becoming an indispensable diagnostic tool for genetic diseases, primarily due to the success of next-generation sequencing (NGS). Large-scale structural variations in the DNA, extending up to megabase pairs in size, that are not identified by NGS can now be captured by long-read genomics technologies such as nanopore sequencing and genome mapping. The merit of the output long-range genomic information obtained from the latter technologies directly depends on the molecular weight distribution of the input DNA sample. The challenge of preventing long DNA molecules from shear-based fragmentation during processing is overcome in conventional sample preparation via macroscopic gel-immobilization of the cells and DNA. This day-long, hands-on method can be tremendously improved by incorporation of microfluidics.

We have developed a poly(dimethylsiloxane) device, inspired by 3D cell culture, to extract high molecular weight DNA from cells in a gel matrix. Rapid DNA purification is achieved by continuous flow in communicating microfluidic channels, which facilitate subsequent electrophoretic extraction of the long DNA out of the miniaturized gel. The disposable PDMS device produces 10 ng DNA from human breast cancer cells at greater than 80% recovery rate. Molecular weight distribution of the device-extracted DNA sample is obtained by molecular combing in microchannels. More than 80% of the characterized DNA molecules are longer than 100 kilobase pairs in size, with the longest molecule extending up to 4 megabase pairs. Our microfluidic cells-to-long DNA process, which is completed in 4 hours, significantly reduces the time, labor and reagent costs associated with long DNA sample preparation. The single-layer microfluidic outlay of the device holds potential for direct integration with genomics chips to obtain a total analysis system.