(H-312) Focused Ultrasound Extraction (FUSE) devices for DNA release from robust tissue matrices

Introduction:: Advancements in DNA sequencing technologies have expanded genomic methods across a variety of disciplines. DNA sequencing is critical to a plethora of applications ranging from biomedical research to plant sciences, but before samples can be sequenced there is often an extensive sample preparation process. Tissue samples must first be homogenized and cells must be lysed to release the DNA, followed by DNA purification and amplification, before the target DNA is ready to be sequenced. For sample types with robust tissue matrices, this preparation process is a limiting step that is labor, time, and resource intensive. Simplifying and automating sample preparation in a cost-effective solution would accelerate genomics research and broaden accessibility to researchers from more backgrounds. To simplify genomic sample preparation workflows, our group is developing focused ultrasound extraction (FUSE) methods for tissue homogenization, cell lysis, and DNA release. FUSE employs high-pressure ultrasound pulses to generate non-thermal, cavitation bubble clouds. The expansion and collapse of the bubble cloud mechanically fragments target tissue into acellular debris, inducing DNA release. The feasibility of FUSE has been characterized for a diverse group of sample types including fresh muscle tissues, formalin-fixed paraffin-embedded (FFPE) tissues, and plant tissues of various mechanical compositions. The challenges associated with genomic sample preparation vary depending on the tissue type, and our results demonstrate that FUSE can overcome many unique, sample-specific obstacles. Overall, feasibility studies suggest that FUSE can simplify genomic sample preparation workflows by rapidly breaking down tissue to release DNA with quality suitable for downstream analyses.




Materials and Methods:: A 32-element, 500 kHz array transducer was used to deliver ultrasound waveforms ranging from 32-40 MPa. Samples were housed in a custom sample holder that suspended tissues in DNA lysis buffer in an optically transparent, acoustically permeable tube. The tube was positioned in the focus of the transducer such that single-cycle ultrasound pulses targeted the tissue samples. Various FUSE processing times were evaluated for all sample types to determine the optimal number of pulses required for efficient tissue pulverization and DNA release. The quantity and quality of DNA released by FUSE were characterized using spectrophotometric and fluorometric methods and compared to conventional DNA extraction methods. The quality of the DNA was further assessed by determining the fragmentation of the DNA released by FUSE. Then the suitability of DNA for downstream analyses was investigated by amplifying the DNA with PCR followed by amplicon or whole genome sequencing.


Results, Conclusions, and Discussions:: The feasibility of FUSE for tissue disintegration and DNA release was determined for piscine muscle tissue, FFPE tissue, leaves, and wood tissue. In all cases, a visual and quantitative inspection of tissues before and after FUSE processing revealed that FUSE was capable of effective tissue disruption. DNA yield results demonstrated that FUSE is capable of releasing quantities of DNA comparable to conventional methods in a fraction of the time. FUSE processing times ranged from 10 seconds to 15 minutes, while conventional methods ranged from 30 minutes to 2 hours. Spectrophotometric assessment of the DNA quality showed that DNA released with FUSE had purity ratios comparable to controls. Visualization of the isolated DNA with gel electrophoresis suggested that FUSE fragments the DNA more than conventional methods for some sample types, dependent upon tissue type and treatment parameters. Amplification results confirmed that FUSE can release high-quality DNA that is suitable for PCR amplification. Lastly, sequencing results demonstrated that samples processed with FUSE can be used for next-generation sequencing applications.

This work determined the feasibility of FUSE technology for releasing DNA suitable for next-generation sequencing applications from a range of sample types. Initial results suggest that FUSE can be developed as a novel sample preparation platform capable of accelerating DNA extraction workflows for a variety of sample types. The improvement of sample throughput has positive implications in fields ranging from fundamental genomics research to medical applications such as diagnostics, personalized medicine, and global health applications. Our ultimate goal is to develop FUSE systems to enable rapid sample processing in field research and point-of-contact settings.


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