Dataset Information

3D Printing-Enabled DNA Extraction for Long-Read Genomics.

ABSTRACT: Long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels extract genomic information in the kilobase to megabase pair range from single DNA molecules, thereby overcoming read-length limitations in next-generation DNA sequencing. Long-read technologies start with long DNA molecules as the input and thus benefit from universal sample preparation methods that are fast and shear-free and present a scope of automation and direct upstream integration. We describe a 3D printing-assisted poly(dimethylysiloxane)-based DNA sample preparation device, where diffusive chemical lysis followed by electrophoresis produces circa 100 ng of long DNA directly from cells with less than 5 min of labor. Assessment of the product DNA by confinement in nanochannels reveals that the DNA sizes are commensurate with the requirements for long-read single-molecule technologies. Microfluidics not only expedites sample preparation, but also offers the opportunity for integration with genomics technologies to eliminate DNA fragmentation and loss during transfer to the genomic device.

SUBMITTER: Agrawal P

PROVIDER: S-EPMC7450497 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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3D Printing-Enabled DNA Extraction for Long-Read Genomics.

Agrawal Paridhi P Reifenberger Jeffrey G JG Dorfman Kevin D KD

ACS omega 20200812 33

Long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels extract genomic information in the kilobase to megabase pair range from single DNA molecules, thereby overcoming read-length limitations in next-generation DNA sequencing. Long-read technologies start with long DNA molecules as the input and thus benefit from universal sample preparation methods that are fast and shear-free and present a scope of automation and direct upstream integration. We describe ...[more]

PMID: 32875216

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Project description:Electro-transfection is an essential workhorse tool for regulating cellular responses and engineering cellular materials in tissue engineering. However, most of the existing approaches are only focused on cell suspensions in vitro, which fails to mimic an in vivo tissue microenvironment regarding the 3D electric field distribution and mass transport in a biological matrix. However, building a 3D electro-transfection system that is compatible with 3D cell culture for mimicking the in vivo tissue microenvironment is challenging, due to the substantial difficulties in control of the 3D electric field distribution as well as the cellular growth. To address such challenges, we introduce a novel 3D micro-assembly strategy assisted by 3D printing, which enables the molding of 3D microstructures as LEGO® parts from 3D-printed molds. The molded PDMS LEGO® bricks are then assembled into a 3D-cell culture chamber interconnected with vertical and horizontal perfusion microchannels as a 3D channel network. Such a 3D perfusion microchannel network is unattainable by direct 3D printing or other microfabrication approaches, which can facilitate the highly-efficient exchange of nutrition and waste for 3D cell growth. Four flat electrodes are mounted into the 3D culture chamber via a 3D-printed holder and controlled by a programmable power sequencer for multi-directional electric frequency scanning (3D ?-electro-transfection). This multi-directional scanning not only can create transient pores all over the cell membrane, but also can generate local oscillation for enhancing mass transport and improving cell transfection efficiency. As a proof-of-concept, we electro-delivered the pAcGFP1-C1 vector to 3D cultured HeLa cells within peptide hydrogel scaffolding. The expressed GFP level from transfected HeLa cells reflects the transfection efficiency. We found two key parameters including electric field strength and plasmid concentration playing more important roles than the pulse duration and duty cycles. The results showed an effective transfection efficiency of ?15% with ?85% cell viability, which is 3-fold higher compared to that of the conventional benchtop 3D cell electro-transfection. This 3D ?-electrotransfection system was further used for genetically editing 3D-cultured Hek-293 cells via direct delivery of CRISPR/Cas9 plasmid which showed successful transfection with GFP expressed in the cytoplasm as the reporter. The 3D-printing enabled micro-assembly allows facile creation of a novel 3D culture system for electro-transfection, which can be employed for versatile gene delivery and cellular engineering, as well as building in vivo like tissue models for fundamentally studying cellular regulation mechanisms at the molecular level.

Project description:Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Dataset Information

3D Printing-Enabled DNA Extraction for Long-Read Genomics.

Publications

3D Printing-Enabled DNA Extraction for Long-Read Genomics.

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